# Downstream bioprocessing of human pluripotent stem cell-derived therapeutics

Sebastien Sart, Chang Liu, Eric Z Zeng, Chunhui Xu, Yan Li

► To cite this version:

Sebastien Sart, Chang Liu, Eric Z Zeng, Chunhui Xu, Yan Li. Downstream bioprocessing of human pluripotent stem cell-derived therapeutics. Engineering in Life Sciences, 2021, 22 (11), pp.667 - 680. 10.1002/elsc.202100042. http://dx.doi.org/10.1002/elsc.202100042

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<figure><img src="image_1.png" /></figure>

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# Downstream bioprocessing of human pluripotent stem cell-derived therapeutics

Sebastien Sart, Chang Liu, Eric Z Zeng, Chunhui Xu, Yan Li

► To cite this version:

Sebastien Sart, Chang Liu, Eric Z Zeng, Chunhui Xu, Yan Li. Downstream bioprocessing of human pluripotent stem cell-derived therapeutics. Engineering in Life Sciences, 2021, 22 (11), pp.667 - 680. 10.1002/elsc.202100042 . hal-04613951

HAL Id: hal-04613951

https://hal.science/hal-04613951v1

Submitted on 17 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# Downstream bioprocessing of human pluripotent stem cell-derived therapeutics

Sebastien Sart<fnref n="1" /> | Chang Liu<fnref n="2" /> | Eric Z. Zeng<fnref n="2" /> | Chunhui Xu<fnref n="3" /> | Yan Li<fnref n="2" />

**Correspondence**

Dr. Sebastien Sart, Laboratory of Physical
Microfluidics and Bioengineering, Depart-
ment of Genome and Genetics, Institut
Pasteur, 75015 Paris, France.

Email: sebastien.sart@gmail.com

Dr. Yan Li, 2525 Pottsdamer St., Tallahassee, FL 32310, USA.

Email: yli@eng.fsu.edu

**Funding information**

National Science Foundation of USA.
Grant/Award Number: 1652992; National
Institutes of Health of USA, Grant/Award
Numbers: R21AA025723, R01HL136345

**Abstract**

With the advancement in lineage-specific differentiation from human pluripo-
tent stem cells (hPSCs), downstream cell separation has now become a critical
step to produce hPSC-derived products. Since differentiation procedures usually
result in a heterogeneous cell population, cell separation needs to be performed
either to enrich the desired cell population or remove the undesired cell popula-
tion. This article summarizes recent advances in separation processes for hPSC-
derived cells, including the standard separation technologies, such as magnetic-
activated cell sorting, as well as the novel separation strategies, such as those
based on adhesion strength and metabolic flux. Specifically, the downstream
bioprocessing flow and the identification of surface markers for various cell lin-
eages are discussed. While challenges remain for large-scale downstream biopro-
cessing of hPSC-derived cells, the rational quality-by-design approach should be
implemented to enhance the understanding of the relationship between process
and the product and to ensure the safety of the produced cells.

**ABBREVIATIONS:** AADAC, arylacetamide deacetylase; AcLDL, acetylated low-density lipoproteins; ALCAM, activated leukocyte cell adhesion molecule; Bim, Bcl-2 interacting mediator of cell death; Cas9, CRISPR-associated protein 9; CD, cluster of differentiation; CLRN3, clarin 3; CNTN2, contactin 2; CRISPR, clustered regularly interspaced short palindromic repeats; cTnT, troponin T; CXCR4, C-X-C motif chemokine receptor 4; DLX2, distal-less homeobox 2; EB, embryoid body; ESC, embryonic stem cells; FACS, fluorescence-activated cell sorting; FOXA2, forkhead box A2; GABA, gamma-aminobutyric acid; GLUT2, glucose transporter 2; HPA, hepatocyte purifying agent; hPSCs, human pluripotent stem cells; ICG, indocyanine green; iPSCs, induced pluripotent stem cells; KDR, kinase insert domain receptor; LMX1A, LIM homeobox transcription factor 1-A; LRTM1, leucine-rich repeats and transmembrane domains 1; MACS, magnetic-activated cell sorting; MBs, molecular beacons; miRNA, microRNA; MLC, myosin regulatory light chain; MYF5, myogenic factor 5; MYH7, myosin heavy chain beta; NCAM, neural cell adhesion molecule; NG2, neural/glial antigen 2; NKX2-5, NK2 homeobox 5; NPCs, neural progenitor cells; NPPA, natriuretic peptide A; NURR1, nuclear receptor related 1 protein; Oct-4, octamer-binding transcription factor 4; OPCs, oligodendrocyte progenitor cells; OSR, odd-skipped related transcription factor; PAX7, paired box 7; PDGFRα, platelet-derived growth factor receptor α; PDX-1, pancreatic and duodenal homeobox 1; PE, pancreatic endoderm; PNIPAAm, poly(N-isopropylacrylamide); QbD, quality by design; ROCK, rho-associated protein kinase; RPE, retinal epithelial cells; SIRPA, signal-regulatory protein alpha; SIX2, SIX homeobox 2; SLC10A1, solute carrier family 10 member 1; SMAD, small mothers against decapentaplegic; SOX, sex determining region Y-box; SSEA, stage-specific embryonic antigen; TALEN, transcription activator-like effector nucleases; TBX5, T-Box transcription factor 5; TH, tyrosine hydroxylase; TH, tyrosine hydroxylase; Tra, tumor-related antigen

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2021 The Authors. *Engineering in Life Sciences* published by Wiley-VCH GmbH

<footnote n="1">¹ Laboratory of Physical Microfluidics and Bioengineering, Department of Genome and Genetics, Institut Pasteur, Paris, France</footnote>

<footnote n="2">² Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA</footnote>

<footnote n="3">³ Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA</footnote>
# Downstream bioprocessing of human pluripotent stem cell-derived therapeutics

Sebastien Sart<fnref n="1" /> | Chang Liu<fnref n="2" /> | Eric Z. Zeng<fnref n="2" /> | Chunhui Xu<fnref n="3" /> | Yan Li<fnref n="2" />

**Correspondence**

Dr. Sebastien Sart, Laboratory of Physical
Microfluidics and Bioengineering, Depart-
ment of Genome and Genetics, Institut
Pasteur, 75015 Paris, France.

Email: sebastien.sart@gmail.com

Dr. Yan Li, 2525 Pottsdamer St., Tallahassee, FL 32310, USA.

Email: yli@eng.fsu.edu

**Funding information**

National Science Foundation of USA.
Grant/Award Number: 1652992; National
Institutes of Health of USA, Grant/Award
Numbers: R21AA025723, R01HL136345

**Abstract**

With the advancement in lineage-specific differentiation from human pluripo-
tent stem cells (hPSCs), downstream cell separation has now become a critical
step to produce hPSC-derived products. Since differentiation procedures usually
result in a heterogeneous cell population, cell separation needs to be performed
either to enrich the desired cell population or remove the undesired cell popula-
tion. This article summarizes recent advances in separation processes for hPSC-
derived cells, including the standard separation technologies, such as magnetic-
activated cell sorting, as well as the novel separation strategies, such as those
based on adhesion strength and metabolic flux. Specifically, the downstream
bioprocessing flow and the identification of surface markers for various cell lin-
eages are discussed. While challenges remain for large-scale downstream biopro-
cessing of hPSC-derived cells, the rational quality-by-design approach should be
implemented to enhance the understanding of the relationship between process
and the product and to ensure the safety of the produced cells.

**ABBREVIATIONS:** AADAC, arylacetamide deacetylase; AcLDL, acetylated low-density lipoproteins; ALCAM, activated leukocyte cell adhesion molecule; Bim, Bcl-2 interacting mediator of cell death; Cas9, CRISPR-associated protein 9; CD, cluster of differentiation; CLRN3, clarin 3; CNTN2, contactin 2; CRISPR, clustered regularly interspaced short palindromic repeats; cTnT, troponin T; CXCR4, C-X-C motif chemokine receptor 4; DLX2, distal-less homeobox 2; EB, embryoid body; ESC, embryonic stem cells; FACS, fluorescence-activated cell sorting; FOXA2, forkhead box A2; GABA, gamma-aminobutyric acid; GLUT2, glucose transporter 2; HPA, hepatocyte purifying agent; hPSCs, human pluripotent stem cells; ICG, indocyanine green; iPSCs, induced pluripotent stem cells; KDR, kinase insert domain receptor; LMX1A, LIM homeobox transcription factor 1-A; LRTM1, leucine-rich repeats and transmembrane domains 1; MACS, magnetic-activated cell sorting; MBs, molecular beacons; miRNA, microRNA; MLC, myosin regulatory light chain; MYF5, myogenic factor 5; MYH7, myosin heavy chain beta; NCAM, neural cell adhesion molecule; NG2, neural/glial antigen 2; NKX2-5, NK2 homeobox 5; NPCs, neural progenitor cells; NPPA, natriuretic peptide A; NURR1, nuclear receptor related 1 protein; Oct-4, octamer-binding transcription factor 4; OPCs, oligodendrocyte progenitor cells; OSR, odd-skipped related transcription factor; PAX7, paired box 7; PDGFRα, platelet-derived growth factor receptor α; PDX-1, pancreatic and duodenal homeobox 1; PE, pancreatic endoderm; PNIPAAm, poly(N-isopropylacrylamide); QbD, quality by design; ROCK, rho-associated protein kinase; RPE, retinal epithelial cells; SIRPA, signal-regulatory protein alpha; SIX2, SIX homeobox 2; SLC10A1, solute carrier family 10 member 1; SMAD, small mothers against decapentaplegic; SOX, sex determining region Y-box; SSEA, stage-specific embryonic antigen; TALEN, transcription activator-like effector nucleases; TBX5, T-Box transcription factor 5; TH, tyrosine hydroxylase; TH, tyrosine hydroxylase; Tra, tumor-related antigen

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2021 The Authors. *Engineering in Life Sciences* published by Wiley-VCH GmbH

<footnote n="1">¹ Laboratory of Physical Microfluidics and Bioengineering, Department of Genome and Genetics, Institut Pasteur, Paris, France</footnote>

<footnote n="2">² Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, FL, USA</footnote>

<footnote n="3">³ Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA</footnote>
<figure><img src="image_2.png" /></figure>

### KEYWORDS

bioprocessing, downstream, pluripotent stem cell, separation

# 1 | INTRODUCTION

Human pluripotent stem cells (hPSCs) provide an alternative cell source for a variety of somatic tissues due to their unique abilities to self-renew and to differentiate into nearly all types of cells. hPSC derivatives have been tested in several phase I clinical trials for their potential use as therapeutic products and also evaluated for drug discovery and disease modeling [1-3]. Initially, the development and manufacturing of hPSC-derived cells have focused on the optimization of differentiation efficiency, i.e. the upstream bioprocessing, leading to improved differentiation protocols that allow the production of lineage-specific cells at high efficiency and purity [3-7]. For examples, oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) can be generated at 70-90% purity from hPSCs using either embryoid body (EB)-based protocol or the monolayer protocol via dual inhibition of SMAD (Small Mothers Against Decapentaplegic) signaling [8, 9], and 30-90% pure cardiomyocytes can be produced using either growth factor- or small molecule-guided protocols [10-12]. The development of such efficient differentiation protocols demonstrates great progress in the upstream bioprocessing for the production of hPSC-derived cells [5]. However, downstream bioprocessing (i.e. cell separation), a critical step and the bottle neck to finalize the hPSC-derived products, requires further investigation [13].

Since current differentiation processes from hPSCs usually result in a mixture of cell types including residual undifferentiated cells [14], downstream bioprocessing needs to be in place for selective purification of desired cell population or removal of unwanted cell populations (Figure 1A). For example, a final stem cell product without undifferentiated cells or progenitors of undesired lineages is critical in order to reduce the risk of teratoma formation after transplantation [14]. Here, we review the emerging downstream bioprocessing for hPSC-derivatives, including recent advances in cell separation after hPSC expansion and differentiation. We provide examples of the identification of novel lineage-specific surface markers, which can be targeted for the separation of hPSC-derived cells. In addition, we discuss methods and practical process flow for hPSC downstream bioprocessing and specific challenges facing the production of neural and cardiac cells.

# 2 | METHODS TO PURIFY HPSC-DERIVED PRODUCTS

During differentiation, hPSCs and derivatives possess stage-specific properties in cell density, expression of surface markers, metabolic requirement, and adhesion strength. Based on these and other characteristics, different types of separation processes for stem cell-derived cells have been evaluated [14], including standard separation technologies such as density-based separation and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) based on specific surface markers. Novel separation strategies, such as cell separation based on differential adherence to culture vessels [15-17] and selective removal of cells based on distinct metabolic activity [18, 19], have also been developed with the better understanding of hPSC properties.

## 2.1 | Density-based separation

A mixture of cell types with different cell densities could be produced in differentiation cultures and may be separated according to their densities. One example is Percoll gradient centrifugation to enrich hPSC-derived cardiomyocytes [20]. Cells harvested from the differentiation culture are loaded onto the two layers of Percoll and then centrifuged. Majority of the cardiomyocytes will be present in the lower layer of Percoll, and non-cardiomyocytes will be in other fractions. Up to 95% pure cardiomyocytes can be obtained from a starting differentiation culture containing about 50% cardiomyocytes [11]. This method, however, has low resolution and is not amenable for large scale separation.

## 2.2 | Separation based on cellular biophysical properties

Substantial differences in biophysical properties among undifferentiated hPSCs, partially reprogrammed cells, somatic cells, and hPSC-derived differentiated cells have been observed [21]. For example, adhesive properties [15], plasmic membrane rigidity [22], and optical characteristics
<figure><img src="image_2.png" /></figure>

### KEYWORDS

bioprocessing, downstream, pluripotent stem cell, separation

# 1 | INTRODUCTION

Human pluripotent stem cells (hPSCs) provide an alternative cell source for a variety of somatic tissues due to their unique abilities to self-renew and to differentiate into nearly all types of cells. hPSC derivatives have been tested in several phase I clinical trials for their potential use as therapeutic products and also evaluated for drug discovery and disease modeling [1–3]. Initially, the development and manufacturing of hPSC-derived cells have focused on the optimization of differentiation efficiency, i.e. the upstream bioprocessing, leading to improved differentiation protocols that allow the production of lineage-specific cells at high efficiency and purity [3–7]. For examples, oligodendrocyte progenitor cells (OPCs) and neural progenitor cells (NPCs) can be generated at 70–90% purity from hPSCs using either embryoid body (EB)-based protocol or the monolayer protocol via dual inhibition of SMAD (Small Mothers Against Decapentaplegic) signaling [8, 9], and 30–90% pure cardiomyocytes can be produced using either growth factor- or small molecule-guided protocols [10–12]. The development of such efficient differentiation protocols demonstrates great progress in the upstream bioprocessing for the production of hPSC-derived cells [5]. However, downstream bioprocessing (i.e. cell separation), a critical step and the bottle neck to finalize the hPSC-derived products, requires further investigation [13].

Since current differentiation processes from hPSCs usually result in a mixture of cell types including residual undifferentiated cells [14], downstream bioprocessing needs to be in place for selective purification of desired cell population or removal of unwanted cell populations (Figure 1A). For example, a final stem cell product without undifferentiated cells or progenitors of undesired lineages is critical in order to reduce the risk of teratoma formation after transplantation [14]. Here, we review the emerging downstream bioprocessing for hPSC-derivatives, including recent advances in cell separation after hPSC expansion and differentiation. We provide examples of the identification of novel lineage-specific surface markers, which can be targeted for the separation of hPSC-derived cells. In addition, we discuss methods and practical process flow for hPSC downstream bioprocessing and specific challenges facing the production of neural and cardiac cells.

# 2 | METHODS TO PURIFY HPSC-DERIVED PRODUCTS

During differentiation, hPSCs and derivatives possess stage-specific properties in cell density, expression of surface markers, metabolic requirement, and adhesion strength. Based on these and other characteristics, different types of separation processes for stem cell-derived cells have been evaluated [14], including standard separation technologies such as density-based separation and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) based on specific surface markers. Novel separation strategies, such as cell separation based on differential adherence to culture vessels [15–17] and selective removal of cells based on distinct metabolic activity [18, 19], have also been developed with the better understanding of hPSC properties.

## 2.1 | Density-based separation

A mixture of cell types with different cell densities could be produced in differentiation cultures and may be separated according to their densities. One example is Percoll gradient centrifugation to enrich hPSC-derived cardiomyocytes [20]. Cells harvested from the differentiation culture are loaded onto the two layers of Percoll and then centrifuged. Majority of the cardiomyocytes will be present in the lower layer of Percoll, and non-cardiomyocytes will be in other fractions. Up to 95% pure cardiomyocytes can be obtained from a starting differentiation culture containing about 50% cardiomyocytes [11]. This method, however, has low resolution and is not amenable for large scale separation.

## 2.2 | Separation based on cellular biophysical properties

Substantial differences in biophysical properties among undifferentiated hPSCs, partially reprogrammed cells, somatic cells, and hPSC-derived differentiated cells have been observed [21]. For example, adhesive properties [15], plasmic membrane rigidity [22], and optical characteristics
<figure><img src="image_3.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>FIGURE 1 Downstream bioprocessing for hPSC-derived products. (A) Enrichment and depletion strategies to isolate hPSC-derived cells. (i) Enrichment: to collect the desired cells (suitable for low differentiation efficiency). (ii) Depletion: to remove undesired cells (suitable for high differentiation efficiency). (B) The major steps of downstream processing. (1) Cell harvesting; (2) centrifugation to remove harvesting enzyme; (3) holding to wait for all the cells to be harvested; (4) depletion to remove the unwanted cells; (5) centrifugation to remove the depletion buffer; and (6) fill and finish to transfer the cells in the cryopreservation buffer for cryopreservation. The overall yield after these steps is expected to be 59% if each step has a 90% yield in an ideal situation. (C) Effects of aggregation on large-scale cell labeling. (i) A single cell suspension allows cell labeling with magnetic beads. (ii) If aggregation happens, the targeted cells cannot be equally labeled and the unlabeled cells in the aggregates are removed, which reduces the yield. (iii) The cells having high aggregation tendency are kept in the formulated buffer as single cell suspension to improve the processing efficiency. This figure contains images from Servier Medical Art (smart.servier.com)</figcaption></figure>

[23] have been shown different at various developmental stages of hPSCs. The changes in these properties can be used as targets for the selection of specific committed populations.

hPSC-derived cardiomyocytes display specific second harmonic signal from myosin rod bundles, which enables their separation from an heterogeneous population of differentiated cells [23]. Alternatively, neural crest cells derived from the replated differentiating hESC aggregates were spontaneously segregated from the main cellular mass, which enable their isolation by shape selection and manual picking [24].

On the other hand, expanded pigmented cells derived from mouse induced pluripotent stem cells (miPSCs) were demonstrated to be more adhesive than non-pigmented cells [15]. This property enables their purification by regulating the time of exposure and the type of enzymatic detachment: non-pigmented colonies can be recovered by a 5-min treatment with Accutase and pipetting, while the remaining adhesive pigmented cells can be secondarily recovered with trypsin [15]. Similarly, medium supplementation with ROCK (Rho-associated protein kinase) inhibitor (Y-27632) prior to cell dissociation, enables the removal of less adhesive cells and thus, to enrich the
<figure><img src="image_3.png" /><figcaption>FIGURE 1 Downstream bioprocessing for hPSC-derived products. (A) Enrichment and depletion strategies to isolate hPSC-derived cells. (i) Enrichment: to collect the desired cells (suitable for low differentiation efficiency). (ii) Depletion: to remove undesired cells (suitable for high differentiation efficiency). (B) The major steps of downstream processing. (1) Cell harvesting; (2) centrifugation to remove harvesting enzyme; (3) holding to wait for all the cells to be harvested; (4) depletion to remove the unwanted cells; (5) centrifugation to remove the depletion buffer; and (6) fill and finish to transfer the cells in the cryopreservation buffer for cryopreservation. The overall yield after these steps is expected to be 59% if each step has a 90% yield in an ideal situation. (C) Effects of aggregation on large-scale cell labeling. (i) A single cell suspension allows cell labeling with magnetic beads. (ii) If aggregation happens, the targeted cells cannot be equally labeled and the unlabeled cells in the aggregates are removed, which reduces the yield. (iii) The cells having high aggregation tendency are kept in the formulated buffer as single cell suspension to improve the processing efficiency. This figure contains images from Servier Medical Art (smart.servier.com)</figcaption></figure>

[23] have been shown different at various developmental stages of hPSCs. The changes in these properties can be used as targets for the selection of specific committed populations.

hPSC-derived cardiomyocytes display specific second harmonic signal from myosin rod bundles, which enables their separation from an heterogeneous population of differentiated cells [23]. Alternatively, neural crest cells derived from the replated differentiating hESC aggregates were spontaneously segregated from the main cellular mass, which enable their isolation by shape selection and manual picking [24].

On the other hand, expanded pigmented cells derived from mouse induced pluripotent stem cells (miPSCs) were demonstrated to be more adhesive than non-pigmented cells [15]. This property enables their purification by regulating the time of exposure and the type of enzymatic detachment: non-pigmented colonies can be recovered by a 5-min treatment with Accutase and pipetting, while the remaining adhesive pigmented cells can be secondarily recovered with trypsin [15]. Similarly, medium supplementation with ROCK (Rho-associated protein kinase) inhibitor (Y-27632) prior to cell dissociation, enables the removal of less adhesive cells and thus, to enrich the
population with endothelial progenitor cells [16]. Moreover, the exit from pluripotency was found to increase cell membrane rigidity. These changes in membrane fluidity and lipid composition can be linked to the differentiation stage. Based on this principle, cell separation can be performed for selecting various differentiated cells as the function of their adhesive properties under membrane fluidization conditions [22].

Human induced pluripotent stem cell (hiPSC) colonies can be detached at a shear stress of 85–125 dynes/cm² within 4 min of fluid-flow application, while the differentiated cells remain attached due to higher adhesive strength. This method resulted in the isolation of fully reprogrammed iPSC colonies to >95% purity from heterogeneous reprogrammed cultures and differentiated progenies. However, this method has not been evaluated for lineage-specific differentiated cells.

Because cell/adhesive surface interactions are regulated upon differentiation, this has led to the development of novel biomaterials for the cell isolation of particular differentiated phenotype [25, 26]. At the undifferentiated stage, iPSC colonies can be selectively detached from thermosensitive polymer (PNIPAAm (poly(N-isopropylacrylamide)), at low temperature (i.e. 22°C), while committed cells remain attached on the surface [25]. This method enables a non-invasive enrichment of undifferentiated stem cell population. Similarly, hiPSCs committed to cardiac lineages seeded on poly(N-isopropylacrylamide) coated with laminin-521, which has a lower critical solution temperature at 8°C, promotes the specific detachment of cardiomyocytes [27]. In addition, it was found that committed cells can be eliminated by a high-speed laser irradiating specific areas of light-responsive polymers (i.e. poly[(methylmethacrylate)-co-(Disperse Yellow 7 methacrylate)] layers) [26]. Alternatively, laminins of different isoforms promote different adhesion strength of various differentiated cell types. For instance, LN211/332/511E8 promotes the adhesion of non-epithelial, while LN332/511E8 favor the attachment and the proliferation of epithelial cells [17]. The specificity of the cell binding to different laminin isoforms has enabled the purification of corneal epithelium cells from heterogeneous differentiated cells [17].

## 2.3 | Selective cell removal based on metabolic activity

Based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and non-cardiomyocytes, high purity (up to 99%) of cardiomyocytes can be obtained from hPSC differentiation culture [18]. The undifferentiated hPSCs and non-cardiac cells that mainly

depend on glycolysis are not able to survive under glucose-depleted and lactate-abundant conditions, whereas cardiomyocytes can survive by using lactate as an alternative energy source. Therefore, cardiomyocytes preferentially survive in glucose-depleted culture medium supplemented with lactate and are consequently enriched. Similarly, using medium depleted with glucose and supplemented with fatty acid and 3,3',5-triiodo-l-thyronine (i.e. a molecule that promotes fatty acid oxidation and mitochondria biogenesis), the selection and the maturation of cardiomyocytes were reported [19].

Retinal epithelial cells (RPE) were found to express high levels of lipoprotein receptors internalize AcLDL (Acetylated Low-Density Lipoproteins). Based on this principle, Dil conjugated-AcLDL was used for the specific labelling and the enrichment of differentiated RPE population [28]. As another example, indocyanine green (ICG) is specifically internalized by hepatocytes. By modifying the fluorescent emission properties of ICG, a hepatocyte purifying agent (HPA, $\lambda_{em}$ = 562 nm) has been designed for the labelling and in vitro sorting of purified hepatocytes derived from hPSCs [29].

## 2.4 | Negative selection

Contrary to positive cell selection, other methods have been developed for the removal of undesired cell types by specifically inducing their apoptosis, thus improving the recovery yield of desired phenotypes. For instance, the transfection of PSCs with the synthetic microRNA (miRNA) switches (i.e. that reduces the translation level of an associated protein in the absence of target miRNA), miR-Bim (Bcl-2 interacting mediator of cell death)-switch, induces the selective apoptosis in undifferentiated cells while maintaining the differentiated cardiomyocytes [30]. Alternatively, the lectin rBC2LCN-PE23 was found to selectively bind and internalize specifically to undifferentiated stem cells and to induce their apoptosis [31]. This compound was found to efficiently reduce the risk of teratoma formation.

## 2.5 | Separation by FACS

The FACS equipment can detect fluorescence signals from cells labeled with fluorochrome-coupled antibodies. Based on the characteristics of labeling and the targeted markers (usually surface antigens), FACS allows sorting and separation of individual cells into different populations (e.g. SSEA (Stage-Specific Embryonic Antigen)-4 positive or SSEA-4 negative). An advantage of FACS is that it allows serial and multi-parametric separation based on multiple
population with endothelial progenitor cells [16]. Moreover, the exit from pluripotency was found to increase cell membrane rigidity. These changes in membrane fluidity and lipid composition can be linked to the differentiation stage. Based on this principle, cell separation can be performed for selecting various differentiated cells as the function of their adhesive properties under membrane fluidization conditions [22].

Human induced pluripotent stem cell (hiPSC) colonies can be detached at a shear stress of 85–125 dynes/cm² within 4 min of fluid-flow application, while the differentiated cells remain attached due to higher adhesive strength. This method resulted in the isolation of fully reprogrammed iPSC colonies to >95% purity from heterogeneous reprogrammed cultures and differentiated progenies. However, this method has not been evaluated for lineage-specific differentiated cells.

Because cell/adhesive surface interactions are regulated upon differentiation, this has led to the development of novel biomaterials for the cell isolation of particular differentiated phenotype [25, 26]. At the undifferentiated stage, iPSC colonies can be selectively detached from thermosensitive polymer (PNIPAAm (poly(N-isopropylacrylamide)), at low temperature (i.e. 22°C), while committed cells remain attached on the surface [25]. This method enables a non-invasive enrichment of undifferentiated stem cell population. Similarly, hiPSCs committed to cardiac lineages seeded on poly(N-isopropylacrylamide) coated with laminin-521, which has a lower critical solution temperature at 8°C, promotes the specific detachment of cardiomyocytes [27]. In addition, it was found that committed cells can be eliminated by a high-speed laser irradiating specific areas of light-responsive polymers (i.e. poly[(methylmethacrylate)-co-(Disperse Yellow 7 methacrylate)] layers) [26]. Alternatively, laminins of different isoforms promote different adhesion strength of various differentiated cell types. For instance, LN211/332/511E8 promotes the adhesion of non-epithelial, while LN332/511E8 favor the attachment and the proliferation of epithelial cells [17]. The specificity of the cell binding to different laminin isoforms has enabled the purification of corneal epithelium cells from heterogeneous differentiated cells [17].

## 2.3 | Selective cell removal based on metabolic activity

Based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and non-cardiomyocytes, high purity (up to 99%) of cardiomyocytes can be obtained from hPSC differentiation culture [18]. The undifferentiated hPSCs and non-cardiac cells that mainly

depend on glycolysis are not able to survive under glucose-depleted and lactate-abundant conditions, whereas cardiomyocytes can survive by using lactate as an alternative energy source. Therefore, cardiomyocytes preferentially survive in glucose-depleted culture medium supplemented with lactate and are consequently enriched. Similarly, using medium depleted with glucose and supplemented with fatty acid and 3,3',5-triiodo-l-thyronine (i.e. a molecule that promotes fatty acid oxidation and mitochondria biogenesis), the selection and the maturation of cardiomyocytes were reported [19].

Retinal epithelial cells (RPE) were found to express high levels of lipoprotein receptors internalize AcLDL (Acetylated Low-Density Lipoproteins). Based on this principle, Dil conjugated-AcLDL was used for the specific labelling and the enrichment of differentiated RPE population [28]. As another example, indocyanine green (ICG) is specifically internalized by hepatocytes. By modifying the fluorescent emission properties of ICG, a hepatocyte purifying agent (HPA, $\lambda_{em}$ = 562 nm) has been designed for the labelling and in vitro sorting of purified hepatocytes derived from hPSCs [29].

## 2.4 | Negative selection

Contrary to positive cell selection, other methods have been developed for the removal of undesired cell types by specifically inducing their apoptosis, thus improving the recovery yield of desired phenotypes. For instance, the transfection of PSCs with the synthetic microRNA (miRNA) switches (i.e. that reduces the translation level of an associated protein in the absence of target miRNA), miR-Bim (Bcl-2 interacting mediator of cell death)-switch, induces the selective apoptosis in undifferentiated cells while maintaining the differentiated cardiomyocytes [30]. Alternatively, the lectin rBC2LCN-PE23 was found to selectively bind and internalize specifically to undifferentiated stem cells and to induce their apoptosis [31]. This compound was found to efficiently reduce the risk of teratoma formation.

## 2.5 | Separation by FACS

The FACS equipment can detect fluorescence signals from cells labeled with fluorochrome-coupled antibodies. Based on the characteristics of labeling and the targeted markers (usually surface antigens), FACS allows sorting and separation of individual cells into different populations (e.g. SSEA (Stage-Specific Embryonic Antigen)-4 positive or SSEA-4 negative). An advantage of FACS is that it allows serial and multi-parametric separation based on multiple
surface markers with high selectivity, although cell viability may be affected by the sorting procedure [32]. However, the throughput of FACS is limited with $5 \times 10^3$ to $7 \times 10^4$ cells sorted per second. Therefore, FACS is more suitable for small-scale cell separation in research use.

## 2.6 | Separation by MACS

MACS is a technique similar to FACS but uses magnetic particles that carry antibodies targeting specific cell surface antigens. In a magnetic field, the magnetically labelled cells will be retained in the column and the unlabelled cells will flow out. For example, cardiomyocytes were enriched to 95% from hPSC differentiation culture by MACS that targeted the cardiomyocyte-associated surface marker VCAM1 (Vascular Cell Adhesion Molecule 1) [33]. However, while MACS is an appealing approach to selectively remove unwanted cells from a cell mixture, it has its limitations. For example, a modeling study suggests that an impractical number of repetitive MACS would be needed to achieve the clearance of undifferentiated stem cells positive for SSEA-1 from a pool of differentiated and undifferentiated cells [34]. However, the affinity of the magnetic beads could also be improved with novel surface modifications to improve sorting efficiency [35]. An advantage of MACS is that it is more practical than FACS for large-scale processing due to the lower cost and the commercial availability of automated, closed-systems [36]. An alternative of MACS beads, the SpheriTECH, enables the purification of photoreceptor progenitor cells derived from hiPSCs without purification label, by simply sorting cells through affinity binding on large beads [37].

## 2.7 | Separation using microfluidics

Microfluidics has recently been demonstrated as efficient tools for hPSC culture, characterization and screening [38]. Microfluidics can also serve for the separation of heterogeneous population of differentiated cells. For instance, microfluidics rolling columns have been fabricated for the selective isolation of SSEA-1⁺ positive cells [39]. The boundaries of the rolling column chips were coated with an antibody that reduced the rolling velocity of the SSEA-1⁺ cells, while committed cells flow out of the channel [39]. Consequently, SSEA-1⁺ cells are retained for longer time within the channel and can consequently be separated from the differentiates cells. Similar operations were performed by covering ridges patterning the floor of microfluidic chips with an anti-Tra (Tumor-related antigen)-1-60 antibody, which eliminates undifferentiated cells from cardiomyocytes derived from hiPSCs [40].

## 3 | SURFACE MARKER IDENTIFICATION FOR THE ISOLATION OF HPSC-DERIVED CELLS

Since FACS and MACS rely on the detection of surface markers, it is important to identify specific surface markers of different lineages during different stages of hPSC differentiation. We discuss examples of studies on surface markers for undifferentiated cells and derivatives of three germ layers including neural cells, cardiomyocytes, and pancreatic progenitors (Table 1).

### 3.1 | Surface markers for undifferentiated cells

Surface markers such as Tra-1-60, Tra-1-81, and SSEA-4 have been used to identify and remove undifferentiated hPSCs (SSEA-1 for mouse embryonic stem cells, mESCs) [41, 42]. For example, FACS and MACS can separate SSEA-4 and Tra-1-81 labeled hESCs (human embryonic stem cells) from other cells [41], and FACS targeting SSEA-5, CD (Cluster of Differentiation)9 and CD90, markers of pluripotent stem cells, can remove cells with teratoma-formation potential from incompletely differentiated hESC cultures [43]. Podocalyxin-like protein-1 is also highly expressed in undifferentiated cells, and a cytotoxic antibody recognizing podocalyxin-like protein-1 has been used to selectively kill undifferentiated hESCs [44]. In addition, specific glycosylation of surface proteins can distinguish pluripotent cells from non-pluripotent cells; therefore, lectins with distinctive binding ability to carbohydrates have been used to remove hPSCs [45].

It should be noted that teratomas could also originate from the precursors that still have stem cell features and are not completely differentiated; thus, these precursors also need to be removed from stem cell-derived products. In any case, for safe cell therapy, it is critical to determine depletion criteria and assay sensitivity of assays to detect undifferentiated stem cells or precursors.

### 3.2 | Surface markers for neural differentiation

Current neural differentiation methods for hPSCs result in cellular heterogeneity with respect to developmental stages and lineage specifications. CD133, A2B5, CD29, CD146, NCAM (Neural Cell Adhesion Molecule) (or CD56) and CD271 were reported to be surface markers on neural progenitor cells (NPCs), and CD24 and NCAM are surface markers for neurons. Targeting CD24
<figure><img src="image_4.png" /></figure>

surface markers with high selectivity, although cell viability may be affected by the sorting procedure [32]. However, the throughput of FACS is limited with $5 \times 10^3$ to $7 \times 10^4$ cells sorted per second. Therefore, FACS is more suitable for small-scale cell separation in research use.

## 2.6 | Separation by MACS

MACS is a technique similar to FACS but uses magnetic particles that carry antibodies targeting specific cell surface antigens. In a magnetic field, the magnetically labelled cells will be retained in the column and the unlabelled cells will flow out. For example, cardiomyocytes were enriched to 95% from hPSC differentiation culture by MACS that targeted the cardiomyocyte-associated surface marker VCAM1 (Vascular Cell Adhesion Molecule 1) [33]. However, while MACS is an appealing approach to selectively remove unwanted cells from a cell mixture, it has its limitations. For example, a modeling study suggests that an impractical number of repetitive MACS would be needed to achieve the clearance of undifferentiated stem cells positive for SSEA-1 from a pool of differentiated and undifferentiated cells [34]. However, the affinity of the magnetic beads could also be improved with novel surface modifications to improve sorting efficiency [35]. An advantage of MACS is that it is more practical than FACS for large-scale processing due to the lower cost and the commercial availability of automated, closed-systems [36]. An alternative of MACS beads, the SpheriTECH, enables the purification of photoreceptor progenitor cells derived from hiPSCs without purification label, by simply sorting cells through affinity binding on large beads [37].

## 2.7 | Separation using microfluidics

Microfluidics has recently been demonstrated as efficient tools for hPSC culture, characterization and screening [38]. Microfluidics can also serve for the separation of heterogeneous population of differentiated cells. For instance, microfluidics rolling columns have been fabricated for the selective isolation of SSEA-1⁺ positive cells [39]. The boundaries of the rolling column chips were coated with an antibody that reduced the rolling velocity of the SSEA-1⁺ cells, while committed cells flow out of the channel [39]. Consequently, SSEA-1⁺ cells are retained for longer time within the channel and can consequently be separated from the differentiates cells. Similar operations were performed by covering ridges patterning the floor of microfluidic chips with an anti-Tra (Tumor-related antigen)-1-60 antibody, which eliminates undifferentiated cells from cardiomyocytes derived from hiPSCs [40].

## 3 | SURFACE MARKER IDENTIFICATION FOR THE ISOLATION OF HPSC-DERIVED CELLS

Since FACS and MACS rely on the detection of surface markers, it is important to identify specific surface markers of different lineages during different stages of hPSC differentiation. We discuss examples of studies on surface markers for undifferentiated cells and derivatives of three germ layers including neural cells, cardiomyocytes, and pancreatic progenitors (Table 1).

### 3.1 | Surface markers for undifferentiated cells

Surface markers such as Tra-1-60, Tra-1-81, and SSEA-4 have been used to identify and remove undifferentiated hPSCs (SSEA-1 for mouse embryonic stem cells, mESCs) [41, 42]. For example, FACS and MACS can separate SSEA-4 and Tra-1-81 labeled hESCs (human embryonic stem cells) from other cells [41], and FACS targeting SSEA-5, CD (Cluster of Differentiation)9 and CD90, markers of pluripotent stem cells, can remove cells with teratoma-formation potential from incompletely differentiated hESC cultures [43]. Podocalyxin-like protein-1 is also highly expressed in undifferentiated cells, and a cytotoxic antibody recognizing podocalyxin-like protein-1 has been used to selectively kill undifferentiated hESCs [44]. In addition, specific glycosylation of surface proteins can distinguish pluripotent cells from non-pluripotent cells; therefore, lectins with distinctive binding ability to carbohydrates have been used to remove hPSCs [45].

It should be noted that teratomas could also originate from the precursors that still have stem cell features and are not completely differentiated; thus, these precursors also need to be removed from stem cell-derived products. In any case, for safe cell therapy, it is critical to determine depletion criteria and assay sensitivity of assays to detect undifferentiated stem cells or precursors.

### 3.2 | Surface markers for neural differentiation

Current neural differentiation methods for hPSCs result in cellular heterogeneity with respect to developmental stages and lineage specifications. CD133, A2B5, CD29, CD146, NCAM (Neural Cell Adhesion Molecule) (or CD56) and CD271 were reported to be surface markers on neural progenitor cells (NPCs), and CD24 and NCAM are surface markers for neurons. Targeting CD24
<table><caption>TABLE 1 Methods used to isolate undifferentiated hPSCs or hPSC-derived cells</caption><thead><tr><td>Isolated cell type</td><td>Methods</td><td>Separation basis</td><td>Separation performance</td><td>Reference</td></tr></thead><tbody><tr><td rowspan="5">Undifferentiated hPSCs</td><td>FACS and MACS</td><td>SSEA-4, Tra-1-60/81</td><td>FACS: all the pluripotent cells removed, lower viability; MACS: 81–84% removed, higher viability</td><td>Fong et al., 2009 [41]</td></tr><tr><td>FACS</td><td>SSEA-5 (with CD9/CD90 or CD50/CD200)</td><td>Teratoma-forming cells completely removed</td><td>Tang et al., 2011 [43]</td></tr><tr><td>Flow shear</td><td>Adhesion strength</td><td>Undifferentiated hPSCs enriched to 95–99%; fast separation (10 min)</td><td>Singh et al., 2013 [21]</td></tr><tr><td>FACS and MACS</td><td>Lectin</td><td>Pluripotent cells removed from mixed populations</td><td>Wang et al., 2011 [45]</td></tr><tr><td>Light response polymer</td><td>Irradiation of differentiated cells</td><td>Purity &gt;98%: TRA-1-60</td><td>Hayashi, et al. 2018 [26]</td></tr><tr><td rowspan="5">hPSC-derived neural progenitors</td><td>FACS and MACS</td><td>Lectin specific binding on undifferentiated cells</td><td>Lectin fusion with a toxin induce apoptosis</td><td>Tateno et al., 2015 [31]</td></tr><tr><td>FACS and MACS</td><td>FORSE-1 NCAM (CD56)</td><td>98% purity for FORSE-1; The isolated NCAM<sup>+</sup> cells had neuronal morphology and express nestin and β-tubulin III</td><td>Pruszak et al., 2007 [32]</td></tr><tr><td>FACS</td><td>CD184<sup>+</sup>/CD271<sup>-</sup>/CD44<sup>-</sup>/CD24<sup>+</sup></td><td>Selection for neural stem cells; CD184<sup>-</sup>/CD44<sup>-</sup>/CD15<sup>low</sup>/CD24<sup>+</sup> for neurons; CD184<sup>+</sup>/CD44<sup>+</sup> for glial cells; &gt;90% purity</td><td>Yuan et al., 2011 [46]</td></tr><tr><td>FACS</td><td>CD15, CD24, CD29</td><td>CD15<sup>+</sup>/CD29<sup>HI</sup>/CD24<sup>LO</sup> defined neural stem cells; CD15<sup>-</sup>/CD29<sup>LO</sup>/CD24<sup>HI</sup> selected neuroblasts and neurons; &gt;95% purity</td><td>Pruszak et al., 2009 [51]</td></tr><tr><td>FACS</td><td>CORIN</td><td>Midbrain dopaminergic progenitors isolated and further differentiated into dopaminergic neurons in vivo.</td><td>Doi et al., 2014 [52]</td></tr><tr><td rowspan="5">hPSC-derived cardiomyocyte progenitors</td><td>FACS</td><td>CD271, CD133</td><td>Purity &gt;80%</td><td>Bowles et al., 2019 [47]</td></tr><tr><td>FACS</td><td>PSA-NCAM, CNTN2</td><td>Purity &gt;80%</td><td>Fathi et al., 2019 [53]</td></tr><tr><td>FACS</td><td>LRTM1</td><td>Purity 30–40%: TH, FOXA2 and NURR1</td><td>Samata et al., 2016 [55]</td></tr><tr><td>FACS</td><td>KDR<sup>low</sup>/C-kit<sup>neg</sup></td><td>Cardiac progenitors isolated and differentiated to cardiomyocytes with &gt;50% purity</td><td>Yang et al., 2008 [59]</td></tr><tr><td>FACS</td><td>KDR<sup>+</sup>/PDGFR-α<sup>+</sup></td><td>Cardiac progenitors isolated and differentiated to cardiomyocytes with &gt;80% purity</td><td>Kattman et al., 2011 [60]</td></tr><tr><td rowspan="3">hPSC-derived cardiomyocytes</td><td>MACS</td><td>VCAM1<sup>+</sup></td><td>95% of cells expressing cardiac troponin T isolated</td><td>Uosaki et al., 2011 [33]</td></tr><tr><td>MACS</td><td>ALCAM1<sup>+</sup></td><td>60% of cardiomyocytes isolated</td><td>Rust et al., 2009 [62]</td></tr><tr><td>FACS</td><td>SIRPA<sup>+</sup></td><td>Up to 98% cardiomyocytes from sources comprising 40–50% cardiomyocytes isolated</td><td>Dubois et al., 2011 [61]</td></tr><tr><td>Fed with glucose-depleted lactate-abundant medium</td><td></td><td>Distinct metabolic flow for different cell types</td><td>Up to 99% pure cardiomyocytes obtained; the preparation did not form tumors after transplantation</td><td>Tohyama et al., 2013 [18]</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 Methods used to isolate undifferentiated hPSCs or hPSC-derived cells</caption><thead><tr><td>Isolated cell type</td><td>Methods</td><td>Separation basis</td><td>Separation performance</td><td>Reference</td></tr></thead><tbody><tr><td rowspan="5">Undifferentiated hPSCs</td><td>FACS and MACS</td><td>SSEA-4, Tra-1-60/81</td><td>FACS: all the pluripotent cells removed, lower viability; MACS: 81–84% removed, higher viability</td><td>Fong et al., 2009 [41]</td></tr><tr><td>FACS</td><td>SSEA-5 (with CD9/CD90 or CD50/CD200)</td><td>Teratoma-forming cells completely removed</td><td>Tang et al., 2011 [43]</td></tr><tr><td>Flow shear</td><td>Adhesion strength</td><td>Undifferentiated hPSCs enriched to 95–99%; fast separation (10 min)</td><td>Singh et al., 2013 [21]</td></tr><tr><td>FACS and MACS</td><td>Lectin</td><td>Pluripotent cells removed from mixed populations</td><td>Wang et al., 2011 [45]</td></tr><tr><td>Light response polymer</td><td>Irradiation of differentiated cells</td><td>Purity &gt;98%: TRA-1-60</td><td>Hayashi, et al. 2018 [26]</td></tr><tr><td rowspan="5">hPSC-derived neural progenitors</td><td>Lectin specific binding on undifferentiated cells</td><td>Lectin fusion with a toxin induce apoptosis</td><td>Remaining undifferentiated cells &lt;0.1%</td><td>Tateno et al., 2015 [31]</td></tr><tr><td>FACS and MACS</td><td>FORSE-1 NCAM (CD56)</td><td>98% purity for FORSE-1; The isolated NCAM<sup>+</sup> cells had neuronal morphology and express nestin and β-tubulin III</td><td>Pruszak et al., 2007 [32]</td></tr><tr><td>FACS</td><td>CD184<sup>+</sup>/CD271<sup>-</sup>/CD44<sup>-</sup>/CD24<sup>+</sup></td><td>Selection for neural stem cells; CD184<sup>-</sup>/CD44<sup>-</sup>/CD15<sup>low</sup>/CD24<sup>+</sup> for neurons; CD184<sup>+</sup>/CD44<sup>+</sup> for glial cells; &gt;90% purity</td><td>Yuan et al., 2011 [46]</td></tr><tr><td>FACS</td><td>CD15, CD24, CD29</td><td>CD15<sup>+</sup>/CD29<sup>HI</sup>/CD24<sup>LO</sup> defined neural stem cells; CD15<sup>-</sup>/CD29<sup>LO</sup>/CD24<sup>HI</sup> selected neuroblasts and neurons; &gt;95% purity</td><td>Pruszak et al., 2009 [51]</td></tr><tr><td>FACS</td><td>CORIN</td><td>Midbrain dopaminergic progenitors isolated and further differentiated into dopaminergic neurons in vivo.</td><td>Doi et al., 2014 [52]</td></tr><tr><td rowspan="5">hPSC-derived cardiomyocyte progenitors</td><td>FACS</td><td>CD271, CD133</td><td>Purity &gt;80%</td><td>Bowles et al., 2019 [47]</td></tr><tr><td>FACS</td><td>PSA-NCAM, CNTN2</td><td>Purity &gt;80%</td><td>Fathi et al., 2019 [53]</td></tr><tr><td>FACS</td><td>LRTM1</td><td>Purity 30–40%: TH, FOXA2 and NURR1</td><td>Samata et al., 2016 [55]</td></tr><tr><td>FACS</td><td>KDR<sup>low</sup>/C-kit<sup>neg</sup></td><td>Cardiac progenitors isolated and differentiated to cardiomyocytes with &gt;50% purity</td><td>Yang et al., 2008 [59]</td></tr><tr><td>FACS</td><td>KDR<sup>+</sup>/PDGFR-α<sup>+</sup></td><td>Cardiac progenitors isolated and differentiated to cardiomyocytes with &gt;80% purity</td><td>Kattman et al., 2011 [60]</td></tr><tr><td rowspan="3">hPSC-derived cardiomyocytes</td><td>MACS</td><td>VCAM1<sup>+</sup></td><td>95% of cells expressing cardiac troponin T isolated</td><td>Uosaki et al., 2011 [33]</td></tr><tr><td>MACS</td><td>ALCAM1<sup>+</sup></td><td>60% of cardiomyocytes isolated</td><td>Rust et al., 2009 [62]</td></tr><tr><td>FACS</td><td>SIRPA<sup>+</sup></td><td>Up to 98% cardiomyocytes from sources comprising 40–50% cardiomyocytes isolated</td><td>Dubois et al., 2011 [61]</td></tr><tr><td>Fed with glucose-depleted lactate-abundant medium</td><td></td><td>Distinct metabolic flow for different cell types</td><td>Up to 99% pure cardiomyocytes obtained; the preparation did not form tumors after transplantation</td><td>Tohyama et al., 2013 [18]</td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><td>Isolated cell type</td><td>Methods</td><td>Separation basis</td><td>Separation performance</td><td>Reference</td></tr></thead><tbody><tr><td></td><td>Percoll gradient centrifugation</td><td>Cell density</td><td>70-95% cardiomyocytes isolated</td><td>Xu et al., 2002 [11, 20]</td></tr><tr><td></td><td>Thermoresponsive polymer</td><td>Adhesion strength</td><td>Purity &gt;90%: TnnT</td><td>Sung et al., 2019 [27]</td></tr><tr><td></td><td>Glucose-depleted medium with fatty acid and 3,3',5-triiodo-l-thyronine</td><td>Distinct metabolic flow for different cell types</td><td>Purity &gt;95% cardiac troponin T</td><td>Lin et al. 2017 [19]</td></tr><tr><td></td><td>miR-Bim-switch</td><td>Distinct miRNA expression for different cell types</td><td>Purity &gt;90%: TnnT</td><td>Miki et al., 2015 [30]</td></tr><tr><td rowspan="3">hPSC-derived pancreatic progenitors</td><td>FACS and MACS</td><td>CD142<sup>-</sup> CD200 and CD318<sup>-</sup></td><td>Pancreatic endoderm (CD142) and endocrine cells separated; FACS: -95% CD142<sup>+</sup> cells; MACS: 75-90% CD142<sup>+</sup> cells</td><td>Kelly et al., 2011 [65]</td></tr><tr><td>FACS</td><td>CD133<sup>+</sup>/CD45<sup>-</sup>/CD34<sup>-</sup></td><td>99% purity obtained that had multi-lineage differentiation capacity</td><td>Oshima et al., 2007 [70]</td></tr><tr><td>FACS and MACS</td><td>GLUT2<sup>-</sup></td><td>FACS: higher purity, lower yield; MACS: lower purity, higher yield.</td><td>Segev et al., 2012 [66]</td></tr><tr><td></td><td>FACS</td><td>CD24<sup>+</sup>NCAM<sup>-</sup></td><td>Purity to &gt;70%, PDX-1<sup>+</sup> progenitors enriched</td><td>Jiang et al., 2011 [67]</td></tr><tr><td>miPSC-derived retinal pigmented cells</td><td>Enzymatic treatment</td><td>Adhesion strength</td><td>Purity &gt;98%</td><td>Iwasaki et al., 2016 [15]</td></tr><tr><td>hiPSC-derived retinal pigmented cells</td><td>Dil conjugated-AcLDL uptake</td><td>Specific molecule uptake</td><td>Pure functional RPE monolayer</td><td>Michelet et al., 2020 [28]</td></tr><tr><td>hiPSC-derived endothelial progenitor cells</td><td>Enzymatic treatment; with Y-27632</td><td>Adhesion strength</td><td>Purity &gt;90%: PECAM1, CDH5, and CD34</td><td>Aoki et al., 2020 [16]</td></tr><tr><td>hiPSC-derived corneal epithelial cells</td><td>Adhesion on different laminin isoform</td><td>Adhesion strength</td><td>Purity ~85%: CD200<sup>-</sup>/SSEA-4<sup>+</sup></td><td>Shibata et al., 2020 [17]</td></tr><tr><td>hPSC-derived hepatocytes</td><td>Hepatocyte purifying agent uptake</td><td>Specific molecule uptake</td><td>Purity &gt;90%: albumin</td><td>Park et al., 2019 [29]</td></tr><tr><td>hiPSC-derived renal progenitor cells</td><td>FACS</td><td>CD9<sup>-</sup>/CD140a<sup>+</sup>/CD140b<sup>+</sup>/CD271<sup>+</sup></td><td>Purity &gt;70%: OSR1, SIX2 and HOXD11</td><td>Hoshina et al., 2018 [73]</td></tr></tbody></table>

or NCAM by FACS enabled the isolation of differentiated neurons [32]. Isolation of NPCs, neurons, and glia cells can also be achieved with combinations of markers (e.g. CD184<sup>+</sup>/CD271<sup>-</sup>/CD44<sup>-</sup>/CD24<sup>+</sup> for NPCs; CD271<sup>-</sup>/CD133<sup>+</sup> for neurons) [46, 47], and different combinations can be used to delineate NPCs: CD184<sup>+</sup>/CD326<sup>-</sup> [48], CD133<sup>+</sup>/CD45<sup>-</sup>/CD34<sup>-</sup> [49], the expression of CD133, CD15, and GCTM-2 [50], and the expression of CD24, CD15, and CD29 [51]. Recently, a floor plate marker CORIN has been used to isolate human iPSC-derived dopaminergic progenitors [52]. Using a LMX1A<sup>EGFP</sup> (LIM homeobox transcription factor 1-A) reporter cell line, novel membrane markers that can be selectively enriched in dopaminergic neurons derived from hESCs have been identified, such as polysialylated embryonic form of neural cell adhe-

sion molecule (PSA-NCAM) and contactin 2 (CNTN2) [53]. Other studies have identified novel surface markers to positively enrich midbrain dopaminergic neurons such as LRTM1 (Leucine-Rich repeats and TransMembrane domains 1), CORIN and CD166, or markers to deplete the undesired cells (CXCR4, C-X-C Motif Chemokine Receptor 4) after the sorting of LMX1<sup>+</sup> FOXA2 (Forkhead Box A2)<sup>+</sup> cells [54, 55].

Surface markers reported for oligodendrocyte progenitors include NG2 (Neural/glial antigen 2), PDGFRα (Platelet-Derived Growth Factor Receptor α), and DLX2 (Distal-Less Homeobox 2) [56] or PDGFRα/CD140 [57]. However, it is difficult to isolate oligodendrocytes based on a single marker because there exists a vast degree of heterogeneity in cellular phenotypes [58].
<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Isolated cell type</th><th>Methods</th><th>Separation basis</th><th>Separation performance</th><th>Reference</th></tr></thead><tbody><tr><td></td><td>Percoll gradient centrifugation</td><td>Cell density</td><td>70-95% cardiomyocytes isolated</td><td>Xu et al., 2002 [11, 20]</td></tr><tr><td></td><td>Thermoresponsive polymer</td><td>Adhesion strength</td><td>Purity &gt;90%: TnnT</td><td>Sung et al., 2019 [27]</td></tr><tr><td></td><td>Glucose-depleted medium with fatty acid and 3,3',5-triiodo-l-thyronine</td><td>Distinct metabolic flow for different cell types</td><td>Purity &gt;95% cardiac troponin T</td><td>Lin et al. 2017 [19]</td></tr><tr><td></td><td>miR-Bim-switch</td><td>Distinct miRNA expression for different cell types</td><td>Purity &gt;90%: TnnT</td><td>Miki et al., 2015 [30]</td></tr><tr><td rowspan="3">hPSC-derived pancreatic progenitors</td><td>FACS and MACS</td><td>CD142<sup>-</sup> CD200 and CD318<sup>-</sup></td><td>Pancreatic endoderm (CD142) and endocrine cells separated; FACS: -95% CD142<sup>+</sup> cells; MACS: 75-90% CD142<sup>+</sup> cells</td><td>Kelly et al., 2011 [65]</td></tr><tr><td>FACS</td><td>CD133<sup>+</sup>/CD45<sup>-</sup>/CD34<sup>-</sup></td><td>99% purity obtained that had multi-lineage differentiation capacity</td><td>Oshima et al., 2007 [70]</td></tr><tr><td>FACS and MACS</td><td>GLUT2<sup>-</sup></td><td>FACS: higher purity, lower yield; MACS: lower purity, higher yield.</td><td>Segev et al., 2012 [66]</td></tr><tr><td></td><td>FACS</td><td>CD24<sup>+</sup>NCAM<sup>-</sup></td><td>Purity to &gt;70%, PDX-1<sup>+</sup> progenitors enriched</td><td>Jiang et al., 2011 [67]</td></tr><tr><td>miPSC-derived retinal pigmented cells</td><td>Enzymatic treatment</td><td>Adhesion strength</td><td>Purity &gt;98%</td><td>Iwasaki et al., 2016 [15]</td></tr><tr><td>hiPSC-derived retinal pigmented cells</td><td>Dil conjugated-AcLDL uptake</td><td>Specific molecule uptake</td><td>Pure functional RPE monolayer</td><td>Michelet et al., 2020 [28]</td></tr><tr><td>hiPSC-derived endothelial progenitor cells</td><td>Enzymatic treatment; with Y-27632</td><td>Adhesion strength</td><td>Purity &gt;90%: PECAM1, CDH5, and CD34</td><td>Aoki et al., 2020 [16]</td></tr><tr><td>hiPSC-derived corneal epithelial cells</td><td>Adhesion on different laminin isoform</td><td>Adhesion strength</td><td>Purity ~85%: CD200<sup>-</sup>/SSEA-4<sup>+</sup></td><td>Shibata et al., 2020 [17]</td></tr><tr><td>hPSC-derived hepatocytes</td><td>Hepatocyte purifying agent uptake</td><td>Specific molecule uptake</td><td>Purity &gt;90%: albumin</td><td>Park et al., 2019 [29]</td></tr><tr><td>hiPSC-derived renal progenitor cells</td><td>FACS</td><td>CD9<sup>-</sup>/CD140a<sup>+</sup>/CD140b<sup>+</sup>/CD271<sup>+</sup></td><td>Purity &gt;70%: OSR1, SIX2 and HOXD11</td><td>Hoshina et al., 2018 [73]</td></tr></tbody></table>

or NCAM by FACS enabled the isolation of differentiated neurons [32]. Isolation of NPCs, neurons, and glia cells can also be achieved with combinations of markers (e.g. CD184<sup>+</sup>/CD271<sup>-</sup>/CD44<sup>-</sup>/CD24<sup>+</sup> for NPCs; CD271<sup>-</sup>/CD133<sup>+</sup> for neurons) [46, 47], and different combinations can be used to delineate NPCs: CD184<sup>+</sup>/CD326<sup>-</sup> [48], CD133<sup>+</sup>/CD45<sup>-</sup>/CD34<sup>-</sup> [49], the expression of CD133, CD15, and GCTM-2 [50], and the expression of CD24, CD15, and CD29 [51]. Recently, a floor plate marker CORIN has been used to isolate human iPSC-derived dopaminergic progenitors [52]. Using a LMX1A<sup>EGFP</sup> (LIM homeobox transcription factor 1-A) reporter cell line, novel membrane markers that can be selectively enriched in dopaminergic neurons derived from hESCs have been identified, such as polysialylated embryonic form of neural cell adhe-

sion molecule (PSA-NCAM) and contactin 2 (CNTN2) [53]. Other studies have identified novel surface markers to positively enrich midbrain dopaminergic neurons such as LRTM1 (Leucine-Rich repeats and TransMembrane domains 1), CORIN and CD166, or markers to deplete the undesired cells (CXCR4, C-X-C Motif Chemokine Receptor 4) after the sorting of LMX1<sup>+</sup> FOXA2 (Forkhead Box A2)<sup>+</sup> cells [54, 55].

Surface markers reported for oligodendrocyte progenitors include NG2 (Neural/glial antigen 2), PDGFRα (Platelet-Derived Growth Factor Receptor α), and DLX2 (Distal-Less Homeobox 2) [56] or PDGFRα/CD140 [57]. However, it is difficult to isolate oligodendrocytes based on a single marker because there exists a vast degree of heterogeneity in cellular phenotypes [58].
## 3.3 | Surface markers for cardiomyocyte differentiation

Markers have been identified both for cardiac progenitor cells and the mature cardiomyocytes. KDR (Kinase insert Domain Receptor)low/C-KITneg cardiac progenitor cells express high levels of cardiac transcription factors and can generate >50% cardiomyocytes after further differentiation [59]. Similarly, the KDR+/PDGFRα+ cardiac progenitor cells can differentiate into >80% cardiomyocytes [60]. In addition, cardiomyocyte-specific surface markers have also been identified, which include signal-regulatory protein alpha (SIRPA), VCAM1, and activated leukocyte cell adhesion molecule (ALCAM) [33, 61, 62]. Following selection based on SIRPA from starting cultures that were ~40-50% cardiac troponin T positive (cTnT+), 90-98% of the cells were positive for cTnT+ [61]. Selection based on VCAM1 by MACS enabled enrichment of cTnT+ cells up to 95% [33]. Compared with these positive selection methods, depletion of unwanted cell population may be considered for cultures with high efficiency of cardiomyocyte differentiation (80-90%). Similarly, cardiomyocytes can be purified using VCAM1-coupled magnetic Dynabeads [63] or integrins (α1, α5, and α6) and N-cadherin to more than 90% purity [64].

## 3.4 | Surface markers for endoderm, hepatic, renal, and pancreatic cell differentiation

hPSC-derived pancreatic cells are heterogeneous. CD142 has been identified as a marker for pancreatic endoderm (PE) cells which give rise to islets with glucose-responsive insulin-secreting cells, and CD200 and CD318 are known as the markers for endocrine cells [65]. Targeting markers such as CD142 can therefore allow enrichment of PE cells, which has been shown to also reduce the probability of teratoma formation (from 46 to 0%) [65]. GLUT2 (Glucose transporter 2), which is co-expressed with PDX-1 (Pancreatic and Duodenal homeobox 1) [66], and CD24, which is shown to correlate with PDX-1+ cells [67], have also been evaluated to enrich pancreatic progenitor cells derived from hPSCs. In addition, surface markers CXCR4, CD49e, CD141 and CD238 have been identified in a study targeting SOX17 to isolate hESC-derived endoderm cells [68, 69]. Although CD133 has been reported as a marker for pancreatic progenitors in mouse, it may have a broader expression other than in pancreatic PDX-1 positive cells in humans [70]. More mature endoderm cells such as hepatocytes derived from PSCs can also be isolate by specific membrane markers (e.g. SLC10A1 (Solute Carrier Family 10 Member 1), CLRN3 (Clarin 3), or AADAC (Arylac-

etamide Deacetylase)) [71], or ASGR1 (i.e. Asialoglycoprotein Receptor 1) [72]. OSR (Odd-Skipped Related Transcription Factor)1+/SIX2 (SIX homeobox 2)+ renal progenitor cells have also been isolated by a set of membrane markers, such as CD9-/-/CD140a+/CD140b+/CD271+ cells [73]. Similarly, corneal epithelial cells can be selected by selectively depleting CD200+ cells [74].

However, questions remain if the cell populations identified by different marker sets are the same cells at different stages or different cells along similar developmental track. For the production purpose, the isolated population needs to be fully characterized and demonstrate the consistent attributes for the targeted applications.

## 3.5 | Non-surface markers for the separation of hPSC-derived cells

Markers other than those expressed on cell membrane but specific to particular cells may be leveraged to isolate these cells. For example, mRNA in live cells can be detected by molecular beacons (MBs) which are stem-loop (hairpin) oligonucleotide probes with a fluorophore at one end and a quencher at the other end [75]. MBs targeting Oct-4 (Octamer-binding transcription factor 4) or Sox2 (Sex determining region Y-box 2) can separate undifferentiated PSCs from the differentiated cells [76, 77]; MBs targeting cardiomyocyte marker myosin heavy chain beta (MYH7) can enrich cardiomyocytes derived from mouse and human embryonic stem cells [78]; and MB targeting NPPA (Natriuretic Peptide A) mRNA, a marker known to be associated with early-stage working-type cardiomyocytes, can enrich working-type cardiomyocyte from cardiomyocyte differentiation culture [79]. For cell separation with MBs, careful design and extensive validation of the MB probes targeting the desired gene are critical.

## 3.6 | Knock-in reporter cell lines

Applications of genome editing technologies (e.g. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), TALEN (transcription activator-like effector nucleases) etc.) have enabled the construction of novel cells lines that are rendered fluorescent upon expression of specific markers or purified with a specific phenotype, through induction of antibiotic resistance. For instance, CRISPR/Cas9 editing of hiPSCs has enabled the generation of double reporter TBX5 (T-Box Transcription Factor 5)Clover2 and NKX2-5 (NK2 Homeobox 5)TagRFP cells that has been used for the purification of heart cell subtypes, such as cells from the first heart field, epicardial, second heart field, and
## 3.3 | Surface markers for cardiomyocyte differentiation

Markers have been identified both for cardiac progenitor cells and the mature cardiomyocytes. KDR (Kinase insert Domain Receptor)low/C-KITneg cardiac progenitor cells express high levels of cardiac transcription factors and can generate >50% cardiomyocytes after further differentiation [59]. Similarly, the KDR+/PDGFRα+ cardiac progenitor cells can differentiate into >80% cardiomyocytes [60]. In addition, cardiomyocyte-specific surface markers have also been identified, which include signal-regulatory protein alpha (SIRPA), VCAM1, and activated leukocyte cell adhesion molecule (ALCAM) [33, 61, 62]. Following selection based on SIRPA from starting cultures that were ~40-50% cardiac troponin T positive (cTnT+), 90-98% of the cells were positive for cTnT+ [61]. Selection based on VCAM1 by MACS enabled enrichment of cTnT+ cells up to 95% [33]. Compared with these positive selection methods, depletion of unwanted cell population may be considered for cultures with high efficiency of cardiomyocyte differentiation (80-90%). Similarly, cardiomyocytes can be purified using VCAM1-coupled magnetic Dynabeads [63] or integrins (α1, α5, and α6) and N-cadherin to more than 90% purity [64].

## 3.4 | Surface markers for endoderm, hepatic, renal, and pancreatic cell differentiation

hPSC-derived pancreatic cells are heterogeneous. CD142 has been identified as a marker for pancreatic endoderm (PE) cells which give rise to islets with glucose-responsive insulin-secreting cells, and CD200 and CD318 are known as the markers for endocrine cells [65]. Targeting markers such as CD142 can therefore allow enrichment of PE cells, which has been shown to also reduce the probability of teratoma formation (from 46 to 0%) [65]. GLUT2 (Glucose transporter 2), which is co-expressed with PDX-1 (Pancreatic and Duodenal homeobox 1) [66], and CD24, which is shown to correlate with PDX-1+ cells [67], have also been evaluated to enrich pancreatic progenitor cells derived from hPSCs. In addition, surface markers CXCR4, CD49e, CD141 and CD238 have been identified in a study targeting SOX17 to isolate hESC-derived endoderm cells [68, 69]. Although CD133 has been reported as a marker for pancreatic progenitors in mouse, it may have a broader expression other than in pancreatic PDX-1 positive cells in humans [70]. More mature endoderm cells such as hepatocytes derived from PSCs can also be isolate by specific membrane markers (e.g. SLC10A1 (Solute Carrier Family 10 Member 1), CLRN3 (Clarin 3), or AADAC (Arylac-

etamide Deacetylase)) [71], or ASGR1 (i.e. Asialoglycoprotein Receptor 1) [72]. OSR (Odd-Skipped Related Transcription Factor)1+/SIX2 (SIX homeobox 2)+ renal progenitor cells have also been isolated by a set of membrane markers, such as CD9-/-/CD140a+/CD140b+/CD271+ cells [73]. Similarly, corneal epithelial cells can be selected by selectively depleting CD200+ cells [74].

However, questions remain if the cell populations identified by different marker sets are the same cells at different stages or different cells along similar developmental track. For the production purpose, the isolated population needs to be fully characterized and demonstrate the consistent attributes for the targeted applications.

## 3.5 | Non-surface markers for the separation of hPSC-derived cells

Markers other than those expressed on cell membrane but specific to particular cells may be leveraged to isolate these cells. For example, mRNA in live cells can be detected by molecular beacons (MBs) which are stem-loop (hairpin) oligonucleotide probes with a fluorophore at one end and a quencher at the other end [75]. MBs targeting Oct-4 (Octamer-binding transcription factor 4) or Sox2 (Sex determining region Y-box 2) can separate undifferentiated PSCs from the differentiated cells [76, 77]; MBs targeting cardiomyocyte marker myosin heavy chain beta (MYH7) can enrich cardiomyocytes derived from mouse and human embryonic stem cells [78]; and MB targeting NPPA (Natriuretic Peptide A) mRNA, a marker known to be associated with early-stage working-type cardiomyocytes, can enrich working-type cardiomyocyte from cardiomyocyte differentiation culture [79]. For cell separation with MBs, careful design and extensive validation of the MB probes targeting the desired gene are critical.

## 3.6 | Knock-in reporter cell lines

Applications of genome editing technologies (e.g. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), TALEN (transcription activator-like effector nucleases) etc.) have enabled the construction of novel cells lines that are rendered fluorescent upon expression of specific markers or purified with a specific phenotype, through induction of antibiotic resistance. For instance, CRISPR/Cas9 editing of hiPSCs has enabled the generation of double reporter TBX5 (T-Box Transcription Factor 5)Clover2 and NKX2-5 (NK2 Homeobox 5)TagRFP cells that has been used for the purification of heart cell subtypes, such as cells from the first heart field, epicardial, second heart field, and
endothelial lineages [80]. Alternatively, NKX2-5<sup>eGFP/w</sup> and MLC (Myosin regulatory Light Chain)2v<sup>mCherry/w</sup> reporter cells have been generated to isolate ventricular like cells [81]. Genome editing (TALEN) have also been used to introduce a selection marker (neomycin) or GFP after the locus of myosin light chain 2 (MYL2) that can serve for identifying and selecting ventricular cardiomyocytes [82]. Similarly, introduction of a Zeocin resistance gene under control of the cardiac-specific α-myosin heavy chain (i.e. α-MHC, MYH6) promoter has been used to purified iPSC-derived cardiomyocytes (iPSC-CMs) in a murine model of myocardial infarction [83].

Similar strategies have been used for the identifi-
cation and the selection of myogenic derivatives. For
instance, a CRISPR/Cas9 mediated homologous recombi-
nation method has been used to select MYF5 (Myogenic
Factor 5)GFP muscle progenitors [84]. The construction of a
double reporter PAX7 (Paired box 7)tdTomato and MYF5EGFP
enabled the purification of muscles stem cells (satellite
cells) [85], while a doxycycline induced PAX7 expression
system promoted the generation of homogeneous popula-
tion of skeletal myogenic progenitors [86].

Reporter cells have also been generated for the purifi-
cation of neural derivatives. For instance, introduction of
a human vesicular GABA (Gamma-Aminobutyric Acid)
transporter (hVGAT) promoter to drive the expression
of mCherry has enable the isolation of GABAergic neu-
rons [87], while midbrain dopaminergic neurons can be
selected using tyrosine hydroxylase (TH)RFP construction
[88]. However, in view of therapeutic applications of hPSC
derivatives, such methods may have increased risk of
tumorigenicity induced by genome modification [89].

4 | WORKFLOW OF DOWNSTREAM BIOPROCESSING FOR HPSC-DERIVED CELLS

Downstream bioprocessing in the production of hPSC-
derived cells involves multiple steps which will accumu-
latively reduce the yield of the process from each step (Fig-
ure 1B) shows an example using MACS depletion). The
major steps are: (1) cell harvesting: dissociation of the cells
from adherent surface of a large amount of culture vessels;
(2) centrifugation or concentration and medium exchange:
to remove harvesting enzyme and wash the cells; (3) hold-
ing: the harvested cells are held in the buffer to wait for
all the cells to be harvested and centrifuged; (4) depletion:
to remove the unwanted impure cells; (5) centrifugation:
to remove the depletion buffer and resuspend in the cry-
opreservation buffer; (6) cell formulation for cryopreser-
vation: to transfer the cells in the cryopreservation buffer
to the cryovials and get ready for cryopreservation. Due to

these many steps, the final yield would be 50–60% if each
step has a yield of 90% (ideal situation) and only 30–35%
if each step has a yield of 80% (good situation). Therefore,
more than half of the cells could be lost due to the down-
stream processing, which usually happens in one work-
ing day after months of cell expansion and differentiation.
Hence, understanding the operation parameters for each
step is definitely required to minimize the cell loss while
maintaining functional viable cells.

4.1 | Cell harvesting

The critical components of cell harvesting from a large number of vessels are the harvesting enzyme and the washing volume. High concentration of enzyme or inappropriate dissociation solutions or duration could reduce cell viability and/or could not remove all the cells on the surface. For differentiation culture, trypsin has been the common enzyme, but its concentration and incubation time need to be optimized for specific cell types. New non-enzymatic passaging method using sodium citrate, which is formulated as a hypertonic solution, has been used to dissociate multicellular aggregates of hPSCs [90]. For bioprocessing, reduced harvesting volume is desired, which requires the balance between low volume and cell loss due to insufficient washing. New advancements in improving the hPSC harvesting have been made by using thermoresponsive hydrogels to release hPSCs by changing the temperature from 37°C to 4°C [91].

4.2 | Centrifugation

The parameters during centrifugation include speed, time, cell suspension volume, and cell concentration. Variations in sedimentation have been observed for different cell collections which trigger multiple centrifugations. The harvesting enzyme, the quenching buffer, and the waiting time may all contribute to the sedimentation performance. For a large number of vessels, development of a large-scale centrifugation process (such as using bags) is required to reduce the time on centrifugation step.

4.3 | Holding buffer and holding temperature

Holding process becomes significant when the number of
vessels is high (>100 T-flasks) and the processing time is
long (as long as 6–8 h). In order to pool all the cells together
for follow-up processing, the cells harvested first have to
sit in the buffer until the harvesting step is completed.
endothelial lineages [80]. Alternatively, NKX2-5<sup>eGFP/w</sup> and MLC (Myosin regulatory Light Chain)2v<sup>mCherry/w</sup> reporter cells have been generated to isolate ventricular like cells [81]. Genome editing (TALEN) have also been used to introduce a selection marker (neomycin) or GFP after the locus of myosin light chain 2 (MYL2) that can serve for identifying and selecting ventricular cardiomyocytes [82]. Similarly, introduction of a Zeocin resistance gene under control of the cardiac-specific α-myosin heavy chain (i.e. α-MHC, MYH6) promoter has been used to purified iPSC-derived cardiomyocytes (iPSC-CMs) in a murine model of myocardial infarction [83].

Similar strategies have been used for the identifi-
cation and the selection of myogenic derivatives. For
instance, a CRISPR/Cas9 mediated homologous recombi-
nation method has been used to select MYF5 (Myogenic
Factor 5)GFP muscle progenitors [84]. The construction of a
double reporter PAX7 (Paired box 7)tdTomato and MYF5EGFP
enabled the purification of muscles stem cells (satellite
cells) [85], while a doxycycline induced PAX7 expression
system promoted the generation of homogeneous popula-
tion of skeletal myogenic progenitors [86].

Reporter cells have also been generated for the purifi-
cation of neural derivatives. For instance, introduction of
a human vesicular GABA (Gamma-Aminobutyric Acid)
transporter (hVGAT) promoter to drive the expression
of mCherry has enable the isolation of GABAergic neu-
rons [87], while midbrain dopaminergic neurons can be
selected using tyrosine hydroxylase (TH)RFP construction
[88]. However, in view of therapeutic applications of hPSC
derivatives, such methods may have increased risk of
tumorigenicity induced by genome modification [89].

4 | WORKFLOW OF DOWNSTREAM BIOPROCESSING FOR HPSC-DERIVED CELLS

Downstream bioprocessing in the production of hPSC-
derived cells involves multiple steps which will accumu-
latively reduce the yield of the process from each step (Fig-
ure 1B) shows an example using MACS depletion). The
major steps are: (1) cell harvesting: dissociation of the cells
from adherent surface of a large amount of culture vessels;
(2) centrifugation or concentration and medium exchange:
to remove harvesting enzyme and wash the cells; (3) hold-
ing: the harvested cells are held in the buffer to wait for
all the cells to be harvested and centrifuged; (4) depletion:
to remove the unwanted impure cells; (5) centrifugation:
to remove the depletion buffer and resuspend in the cry-
opreservation buffer; (6) cell formulation for cryopreser-
vation: to transfer the cells in the cryopreservation buffer
to the cryovials and get ready for cryopreservation. Due to

these many steps, the final yield would be 50-60% if each
step has a yield of 90% (ideal situation) and only 30-35%
if each step has a yield of 80% (good situation). Therefore,
more than half of the cells could be lost due to the down-
stream processing, which usually happens in one work-
ing day after months of cell expansion and differentiation.
Hence, understanding the operation parameters for each
step is definitely required to minimize the cell loss while
maintaining functional viable cells.

**4.1** | **Cell harvesting**

The critical components of cell harvesting from a large number of vessels are the harvesting enzyme and the washing volume. High concentration of enzyme or inappropriate dissociation solutions or duration could reduce cell viability and/or could not remove all the cells on the surface. For differentiation culture, trypsin has been the common enzyme, but its concentration and incubation time need to be optimized for specific cell types. New non-enzymatic passaging method using sodium citrate, which is formulated as a hypertonic solution, has been used to dissociate multicellular aggregates of hPSCs [90]. For bioprocessing, reduced harvesting volume is desired, which requires the balance between low volume and cell loss due to insufficient washing. New advancements in improving the hPSC harvesting have been made by using thermoresponsive hydrogels to release hPSCs by changing the temperature from 37°C to 4°C [91].

4.2 | Centrifugation

The parameters during centrifugation include speed, time, cell suspension volume, and cell concentration. Variations in sedimentation have been observed for different cell collections which trigger multiple centrifugations. The harvesting enzyme, the quenching buffer, and the waiting time may all contribute to the sedimentation performance. For a large number of vessels, development of a large-scale centrifugation process (such as using bags) is required to reduce the time on centrifugation step.

4.3 | Holding buffer and holding temperature

Holding process becomes significant when the number of
vessels is high (>100 T-flasks) and the processing time is
long (as long as 6-8 h). In order to pool all the cells together
for follow-up processing, the cells harvested first have to
sit in the buffer until the harvesting step is completed.
Hence, the holding buffer and the holding temperature (37°C, 25°C, or 4°C) need to be evaluated to maintain cell viability. To formulate the holding buffer, one should take consideration of cell aggregation. Although the harvested cells are in single cell suspension, the cells may self-assemble into aggregates during the holding period. The degree of aggregation depends on cell type, cell concentration, and holding time. The formation of aggregates will affect the efficiency of cell labeling and separation in subsequent steps, significantly reducing cell yield.

## 4.4 | Depletion buffer formulation and volume

Depletion is applied to remove the small number of impure cells when the population has a high percentage of desired cells. This process can be done by MACS targeting surface markers on the undesired cells. The requirement for depletion buffer is similar to holding buffer: maintaining cell viability and preventing cell aggregation. The aggregated cells do not allow homogeneous labeling with the antibody-conjugated beads and easy cell-bead separation (Figure 1C). Consequently, the depletion buffer needs to be carefully formulated. The cell concentration and the volume for depletion affect the depletion scale. High cell concentration (>10⁷ cells/mL) and low volume (<100 mL) are preferred, while cell loss needs to be minimized. Since multiple markers could be used for depletion, this step may be repeated to remove different cell populations.

## 4.5 | Formulation for cryopreservation

Cryopreservation buffer and volume are the important parameters of this step. In general, the cryopreservation buffer depends on the differentiated cell types of interest, and the cryopreserved cell concentration depends on the intended applications. For transplantation studies, high concentration of cells (10⁷-10⁸ cells per vial) may be required to minimize the time for thawing process and the number of vials to achieve the desired dose of cells.

# 5 | CHALLENGES IN DOWNSTREAM PROCESSING HPSC-DERIVED PRODUCTS

## 5.1 | Challenges in downstream processing for hPSC-derived neural progenitor cells

Large-scale purification of neurons derived from PSCs has been achieved using MACS, yielding to the recovery of

up to 10⁵–10⁶ neurons (more than 90% purity) [92]. However, specific challenges remain for downstream processing of these specific lineages. In the case of neural progenitor cells, the harvesting cell density is relatively low (about 1 × 10⁶ cells per cm²), which could require to harvest cells from a large number of vessels. If the higher harvesting density can be reached, the number of required vessels will be significantly reduced to obtain the desired number of cells (~10⁹ cells per production, 50–100 mL downstream processing volume). Increasing the harvesting cell density may be achieved by increasing seeding density and/or enhancing cell proliferation. However, depending on the culture system and the sensitivity of cell phenotype to the density change, seeding density may affect the secretion of autocrine and paracrine factors which have been shown to affect hPSC self-renewal and differentiation [93]. To enhance cell proliferation, one could consider changing growth medium (containing growth factors) and the substrates which are usually coated with matrix proteins such as laminin, Matrigel, and fibronectin. However, any changes made in media and substrates need to be carefully compared.

## 5.2 | Challenges in downstream processing for hPSC-derived cardiomyocytes

For cardiomyocytes, the burden on the downstream bioprocessing is more severe than neural progenitors. The quantity of cells needed for therapeutic applications is higher for cardiomyocytes (~10¹⁰ cells per production) than for neural progenitors (~10⁹ per production) [5]. For all the steps in downstream bioprocessing of cardiomyocytes, it may require 10-times more concentrated cell preparation or 10-times more suspension volume compared with that of neural progenitors. Large-scale purification of cardiomyocytes derived from human PSCs has been achieved using metabolic selection methods reaching 99% purity and yielding to the recovery of up to 2 × 10⁹ cells functional cells [18, 94, 95]. Cardiomyocytes also form aggregates more easily than neural progenitors and thus the yield of the depletion step could be lower. To efficiently label the cells with magnetic beads and separate different populations, cardiomyocytes need to be maintained as single cell suspension during downstream bioprocessing. Thus, the holding buffer and depletion buffer need to be formulated (e.g. anti-clumping agents, biopolymers) to prevent cell clumping (Figure 1C). The scale of downstream processing of hPSC-derived cardiomyocytes could be large given the quantity needed for therapeutic use. Therefore, scale-up of different steps during downstream bioprocessing will be required (e.g.
Hence, the holding buffer and the holding temperature (37°C, 25°C, or 4°C) need to be evaluated to maintain cell viability. To formulate the holding buffer, one should take consideration of cell aggregation. Although the harvested cells are in single cell suspension, the cells may self-assemble into aggregates during the holding period. The degree of aggregation depends on cell type, cell concentration, and holding time. The formation of aggregates will affect the efficiency of cell labeling and separation in subsequent steps, significantly reducing cell yield.

## 4.4 | Depletion buffer formulation and volume

Depletion is applied to remove the small number of impure cells when the population has a high percentage of desired cells. This process can be done by MACS targeting surface markers on the undesired cells. The requirement for depletion buffer is similar to holding buffer: maintaining cell viability and preventing cell aggregation. The aggregated cells do not allow homogeneous labeling with the antibody-conjugated beads and easy cell-bead separation (Figure 1C). Consequently, the depletion buffer needs to be carefully formulated. The cell concentration and the volume for depletion affect the depletion scale. High cell concentration (>10⁷ cells/mL) and low volume (<100 mL) are preferred, while cell loss needs to be minimized. Since multiple markers could be used for depletion, this step may be repeated to remove different cell populations.

## 4.5 | Formulation for cryopreservation

Cryopreservation buffer and volume are the important parameters of this step. In general, the cryopreservation buffer depends on the differentiated cell types of interest, and the cryopreserved cell concentration depends on the intended applications. For transplantation studies, high concentration of cells (10⁷-10⁸ cells per vial) may be required to minimize the time for thawing process and the number of vials to achieve the desired dose of cells.

# 5 | CHALLENGES IN DOWNSTREAM PROCESSING HPSC-DERIVED PRODUCTS

## 5.1 | Challenges in downstream processing for hPSC-derived neural progenitor cells

Large-scale purification of neurons derived from PSCs has been achieved using MACS, yielding to the recovery of

up to 10⁵–10⁶ neurons (more than 90% purity) [92]. However, specific challenges remain for downstream processing of these specific lineages. In the case of neural progenitor cells, the harvesting cell density is relatively low (about 1 × 10⁶ cells per cm²), which could require to harvest cells from a large number of vessels. If the higher harvesting density can be reached, the number of required vessels will be significantly reduced to obtain the desired number of cells (~10⁹ cells per production, 50–100 mL downstream processing volume). Increasing the harvesting cell density may be achieved by increasing seeding density and/or enhancing cell proliferation. However, depending on the culture system and the sensitivity of cell phenotype to the density change, seeding density may affect the secretion of autocrine and paracrine factors which have been shown to affect hPSC self-renewal and differentiation [93]. To enhance cell proliferation, one could consider changing growth medium (containing growth factors) and the substrates which are usually coated with matrix proteins such as laminin, Matrigel, and fibronectin. However, any changes made in media and substrates need to be carefully compared.

## 5.2 | Challenges in downstream processing for hPSC-derived cardiomyocytes

For cardiomyocytes, the burden on the downstream bioprocessing is more severe than neural progenitors. The quantity of cells needed for therapeutic applications is higher for cardiomyocytes (~10¹⁰ cells per production) than for neural progenitors (~10⁹ per production) [5]. For all the steps in downstream bioprocessing of cardiomyocytes, it may require 10-times more concentrated cell preparation or 10-times more suspension volume compared with that of neural progenitors. Large-scale purification of cardiomyocytes derived from human PSCs has been achieved using metabolic selection methods reaching 99% purity and yielding to the recovery of up to 2 × 10⁹ cells functional cells [18, 94, 95]. Cardiomyocytes also form aggregates more easily than neural progenitors and thus the yield of the depletion step could be lower. To efficiently label the cells with magnetic beads and separate different populations, cardiomyocytes need to be maintained as single cell suspension during downstream bioprocessing. Thus, the holding buffer and depletion buffer need to be formulated (e.g. anti-clumping agents, biopolymers) to prevent cell clumping (Figure 1C). The scale of downstream processing of hPSC-derived cardiomyocytes could be large given the quantity needed for therapeutic use. Therefore, scale-up of different steps during downstream bioprocessing will be required (e.g.
21. Singh, A., Suri, S., Lee, T., Chilton, J. M., et al. Adhesion strength-based, label-free isolation of human pluripotent stem cells. Nat. Methods 2013, 10, 438–444.

22. Matsuzaki, T., Matsumoto, S., Kasai, T., Yoshizawa, E., et al. Defining lineage-specific membrane fluidity signatures that regulate adhesion kinetics. Stem Cell Reports 2018, 11, 852–860.

23. Chang, C. W., Kao, H. K. J., Yechikov, S., Lieu, D. K., et al. An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype. Stem Cells 2020, 38, 390–394.

24. Munst, S., Koch, P., Kesavan, J., Alexander-Mays, M., et al. In vitro segregation and isolation of human pluripotent stem cell-derived neural crest cells, Methods 2018, 133, 65–80.

25. Jiang, S., Muller, M., Schonherr, H., Propagation and purification of human induced pluripotent stem cells with selective homopolymer release surfaces. Angew. Chem. Int. Ed. Engl. 2019, 58, 10563–10566.

26. Hayashi, Y., Matsumoto, J., Kumagai, S., Morishita, K., et al. Automated adherent cell elimination by a high-speed laser mediated by a light-responsive polymer. Commun Biol 2018, 1, 218.

27. Sung, T. C., Su, H. C., Ling, Q. D., Kumar, S. S., et al. Efficient differentiation of human pluripotent stem cells into cardiomyocytes on cell sorting thermoresponsive surface. Biomaterials 2020, 253, 120060.

28. Michelet, F., Balasankar, A., Teo, N., Stanton, L. W., et al. Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting. Stem Cell. Res. Ther. 2020, 11, 47.

29. Park, J. Y., Han, J., Jung, H. S., Lee, G., et al. Synthetic probes for in vitro purification and in vivo tracking of hepatocytes derived from human pluripotent stem cells. Biomaterials 2019, 222, 119431.

30. Miki, K., Endo, K., Takahashi, S., Funakoshi, S., et al. Efficient detection and purification of cell populations using synthetic microRNA switches. Cell Stem Cell 2015, 16, 699–711.

31. Tateno, H., Onuma, Y., Ito, Y., Minoshima, F., et al. Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein. Stem Cell Reports 2015, 4, 811–820.

32. Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., et al. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 2007, 25, 2257–2268.

33. Uosaki, H., Fukushima, H., Takeuchi, A., Matsuoka, S., et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression, PLoS One 2011, 6, e23657.

34. Schriebl, K., Lim, S., Choo, A., Tscheliessnig, A., et al. Stem cell separation: a bottleneck in stem cell therapy. Biotechnol. J. 2010, 5, 50–61.

35. Borlido, L., Azevedo, A. M., Roque, A. C., Aires-Barros, M. R., Magnetic separations in biotechnology. Biotechnol. Adv. 2013, 31, 1374–1385.

36. Li, Y., Green, M., Wen, Y., Wei, Y., et al. Efficacy and safety of immuno-magnetically sorted smooth muscle progenitor cells derived from human-induced pluripotent stem cells for restoring urethral sphincter function, Stem Cells Transl Med 2017, 6, 1158–1167.

37. Weil, B. D., Jenkins, M. J., Uddin, S., Bracewell, D. G., et al. An integrated experimental and economic evaluation of cell ther-

apy affinity purification technologies. Regen. Med. 2017, 12, 397–417.

38. Sart, S., Agathos, S. N., Towards three-dimensional dynamic regulation and in situ characterization of single stem cell phenotype using microfluidics. Mol. Biotechnol. 2018, 60, 843–861.

39. Otaka, A., Kitagawa, K., Nakaoki, T., Hirata, M., et al. Label-free separation of induced pluripotent stem cells with anti-SSEA-1 Antibody immobilized microfluidic channel. Langmuir 2017, 33, 1576–1582.

40. Li, X., Yu, L., Li, J., Minami, I., et al. On chip purification of hiPSC-derived cardiomyocytes using a fishnet-like microstructure. Biofabrication 2016, 8, 035017.

41. Fong, C. Y., Peh, G. S., Gauthaman, K., Bongso, A., Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Stem cell rev. 2009, 5, 72–80.

42. Shibata, H., Ageyama, N., Tanaka, Y., Kishi, Y., et al. Improved safety of hematopoietic transplantation with monkey embryonic stem cells in the allogeneic setting. Stem Cells 2006, 24, 1450–1457.

43. Tang, C., Lee, A. S., Volkmer, J. P., Sahoo, D., et al. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat. Biotechnol. 2011, 29, 829–834.

44. Choo, A. B., Tan, H. L., Ang, S. N., Fong, W. J., et al. Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podalyxin-like protein-1. Stem Cells 2008, 26, 1454–1463.

45. Wang, Y. C., Nakagawa, M., Garitaonandia, I., Slavin, I., et al. Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomics analysis. Cell Res. 2011, 21, 1551–1563.

46. Yuan, S. H., Martin, J., Elia, J., Flippin, J., et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 2011, 6, e17540.

47. Bowles, K. R., Tcw, J., Qian, L., Jadow, B. M., et al. Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. PLoS One 2019, 14, e0213374.

48. Sundberg, M., Jansson, L., Ketolainen, J., Pihlajamaki, H., et al. CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res. 2009, 2, 113–124.

49. Golebiewska, A., Atkinson, S. P., Lako, M., Armstrong, L., Epigenetic landscaping during hESC differentiation to neural cells, Stem Cells 2009, 27, 1298–1308.

50. Peh, G. S., Lang, R. J., Pera, M. F., Hawes, S. M., CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation. Stem Cells Dev. 2009, 18, 269–282.

51. Pruszak, J., Ludwig, W., Blak, A., Alavian, K., et al. CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells. Stem Cells 2009, 27, 2928–2940.

52. Doi, D., Samata, B., Katsukawa, M., Kikuchi, T., et al. Isolation of human induced pluripotent stem cell-derived dopaminergic
21. Singh, A., Suri, S., Lee, T., Chilton, J. M., et al. Adhesion strength-based, label-free isolation of human pluripotent stem cells. Nat. Methods 2013, 10, 438–444.

22. Matsuzaki, T., Matsumoto, S., Kasai, T., Yoshizawa, E., et al. Defining lineage-specific membrane fluidity signatures that regulate adhesion kinetics. Stem Cell Reports 2018, 11, 852–860.

23. Chang, C. W., Kao, H. K. J., Yechikov, S., Lieu, D. K., et al. An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype. Stem Cells 2020, 38, 390–394.

24. Munst, S., Koch, P., Kesavan, J., Alexander-Mays, M., et al. In vitro segregation and isolation of human pluripotent stem cell-derived neural crest cells, Methods 2018, 133, 65–80.

25. Jiang, S., Muller, M., Schonherr, H., Propagation and purification of human induced pluripotent stem cells with selective homopolymer release surfaces. Angew. Chem. Int. Ed. Engl. 2019, 58, 10563–10566.

26. Hayashi, Y., Matsumoto, J., Kumagai, S., Morishita, K., et al. Automated adherent cell elimination by a high-speed laser mediated by a light-responsive polymer. Commun Biol 2018, 1, 218.

27. Sung, T. C., Su, H. C., Ling, Q. D., Kumar, S. S., et al. Efficient differentiation of human pluripotent stem cells into cardiomyocytes on cell sorting thermoresponsive surface. Biomaterials 2020, 253, 120060.

28. Michelet, F., Balasankar, A., Teo, N., Stanton, L. W., et al. Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting. Stem Cell. Res. Ther. 2020, 11, 47.

29. Park, J. Y., Han, J., Jung, H. S., Lee, G., et al. Synthetic probes for in vitro purification and in vivo tracking of hepatocytes derived from human pluripotent stem cells. Biomaterials 2019, 222, 119431.

30. Miki, K., Endo, K., Takahashi, S., Funakoshi, S., et al. Efficient detection and purification of cell populations using synthetic microRNA switches. Cell Stem Cell 2015, 16, 699–711.

31. Tateno, H., Onuma, Y., Ito, Y., Minoshima, F., et al. Elimination of tumorigenic human pluripotent stem cells by a recombinant lectin-toxin fusion protein. Stem Cell Reports 2015, 4, 811–820.

32. Pruszak, J., Sonntag, K. C., Aung, M. H., Sanchez-Pernaute, R., et al. Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations. Stem Cells 2007, 25, 2257–2268.

33. Uosaki, H., Fukushima, H., Takeuchi, A., Matsuoka, S., et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression, PLoS One 2011, 6, e23657.

34. Schriebl, K., Lim, S., Choo, A., Tscheliessnig, A., et al. Stem cell separation: a bottleneck in stem cell therapy. Biotechnol. J. 2010, 5, 50–61.

35. Borlido, L., Azevedo, A. M., Roque, A. C., Aires-Barros, M. R., Magnetic separations in biotechnology. Biotechnol. Adv. 2013, 31, 1374–1385.

36. Li, Y., Green, M., Wen, Y., Wei, Y., et al. Efficacy and safety of immuno-magnetically sorted smooth muscle progenitor cells derived from human-induced pluripotent stem cells for restoring urethral sphincter function, Stem Cells Transl Med 2017, 6, 1158–1167.

37. Weil, B. D., Jenkins, M. J., Uddin, S., Bracewell, D. G., et al. An integrated experimental and economic evaluation of cell ther-

apy affinity purification technologies. Regen. Med. 2017, 12, 397–417.

38. Sart, S., Agathos, S. N., Towards three-dimensional dynamic regulation and in situ characterization of single stem cell phenotype using microfluidics. Mol. Biotechnol. 2018, 60, 843–861.

39. Otaka, A., Kitagawa, K., Nakaoki, T., Hirata, M., et al. Label-free separation of induced pluripotent stem cells with anti-SSEA-1 Antibody immobilized microfluidic channel. Langmuir 2017, 33, 1576–1582.

40. Li, X., Yu, L., Li, J., Minami, I., et al. On chip purification of hiPSC-derived cardiomyocytes using a fishnet-like microstructure. Biofabrication 2016, 8, 035017.

41. Fong, C. Y., Peh, G. S., Gauthaman, K., Bongso, A., Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Stem cell rev. 2009, 5, 72–80.

42. Shibata, H., Ageyama, N., Tanaka, Y., Kishi, Y., et al. Improved safety of hematopoietic transplantation with monkey embryonic stem cells in the allogeneic setting. Stem Cells 2006, 24, 1450–1457.

43. Tang, C., Lee, A. S., Volkmer, J. P., Sahoo, D., et al. An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells. Nat. Biotechnol. 2011, 29, 829–834.

44. Choo, A. B., Tan, H. L., Ang, S. N., Fong, W. J., et al. Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podalyxin-like protein-1. Stem Cells 2008, 26, 1454–1463.

45. Wang, Y. C., Nakagawa, M., Garitaonandia, I., Slavin, I., et al. Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomics analysis. Cell Res. 2011, 21, 1551–1563.

46. Yuan, S. H., Martin, J., Elia, J., Flippin, J., et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 2011, 6, e17540.

47. Bowles, K. R., Tcw, J., Qian, L., Jadow, B. M., et al. Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. PLoS One 2019, 14, e0213374.

48. Sundberg, M., Jansson, L., Ketolainen, J., Pihlajamaki, H., et al. CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells. Stem Cell Res. 2009, 2, 113–124.

49. Golebiewska, A., Atkinson, S. P., Lako, M., Armstrong, L., Epigenetic landscaping during hESC differentiation to neural cells, Stem Cells 2009, 27, 1298–1308.

50. Peh, G. S., Lang, R. J., Pera, M. F., Hawes, S. M., CD133 expression by neural progenitors derived from human embryonic stem cells and its use for their prospective isolation. Stem Cells Dev. 2009, 18, 269–282.

51. Pruszak, J., Ludwig, W., Blak, A., Alavian, K., et al. CD15, CD24, and CD29 define a surface biomarker code for neural lineage differentiation of stem cells. Stem Cells 2009, 27, 2928–2940.

52. Doi, D., Samata, B., Katsukawa, M., Kikuchi, T., et al. Isolation of human induced pluripotent stem cell-derived dopaminergic
progenitors by cell sorting for successful transplantation. Stem cell reports 2014, 2, 337–350.

53. Fathi, A., Mirzaei, M., Dolatyar, B., Sharifitabar, M., et al. Discovery of novel cell surface markers for purification of embryonic dopamine progenitors for transplantation in Parkinson's disease animal models. Mol. Cell. Proteomics 2018, 17, 1670–1684.

54. Paik, E. J., O'Neil, A. L., Ng, S. Y., Sun, C., et al. Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hiPSC culture. Sci. Rep. 2018, 8, 804.

55. Samata, B., Doi, D., Nishimura, K., Kikuchi, T., et al. Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1. Nat Commun 2016, 7, 13097.

56. Sundberg, M., Skottman, H., Suuronen, R., Narkilahti, S., Production and isolation of NG2+ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium. Stem cell res. 2010, 5, 91–103.

57. Djelloul, M., Azevedo, C., Pomeshchik, Y., Hammarberg, A., et al. Reporting on methods to generate and purify rodent and human oligodendrocytes from different sources. Stem Cell Res 2017, 20, 58–66.

58. Alsanie, W. F., Niclis, J. C., Petratos, S., Human embryonic stem cell-derived oligodendrocytes: protocols and perspectives. Stem Cells Dev. 2013, 22, 2459–2476.

59. Yang, L., Soonpaa, M. H., Adler, E. D., Roepke, T. K., et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 2008, 453, 524–528.

60. Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 2011, 8, 228–240.

61. Dubois, N. C., Craft, A. M., Sharma, P., Elliott, D. A., et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 2011, 29, 1011–1018.

62. Rust, W., Balakrishnan, T., Zweigerdt, R., Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression. Regen. Med. 2009, 4, 225–237.

63. Schwach, V., Passier, R., Generation and purification of human stem cell-derived cardiomyocytes. Differentiation 2016, 91, 126–138.

64. Tarnawski, L., Xian, X., Monnerat, G., Macaulay, I. C., et al. Integrin based isolation enables purification of murine lineage committed cardiomyocytes. PLoS One 2015, 10, e0135880.

65. Kelly, O. G., Chan, M. Y., Martinson, L. A., Kadoya, K., et al. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat. Biotechnol. 2011, 29, 750–756.

66. Segev, H., Fishman, B., Schulman, R., Itskovitz-Eldor, J., The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment. Stem Cells Dev 2012, 21, 1653–1661.

67. Jiang, W., Sui, X., Zhang, D., Liu, M., et al. CD24: a novel surface marker for PDX1-positive pancreatic progenitors derived from human embryonic stem cells. Stem Cells 2011, 29, 609–617.

68. Wang, P., Rodriguez, R. T., Wang, J., Ghodasara, A., et al. Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell 2011, 8, 335–346.

69. Davenport, C., Diekmann, U., Naujok, O., A quick and efficient method for the purification of endoderm cells generated from human embryonic stem cells. J Vis Exp 2016.

70. Oshima, Y., Suzuki, A., Kawashimo, K., Ishikawa, M., et al. Isolation of mouse pancreatic ductal progenitor cells expressing CD133 and c-Met by flow cytometric cell sorting. Gastroenterology 2007, 132, 720–732.

71. Mallanna, S. K., Cayo, M. A., Twaroski, K., Gundry, R. L., et al. Mapping the cell-surface N-glycoproteome of human hepatocytes reveals markers for selecting a homogeneous population of iPSC-derived hepatocytes. Stem Cell Reports 2016, 7, 543–556.

72. Peters, D. T., Henderson, C. A., Warren, C. R., Friesen, M., et al. Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells. Development 2016, 143, 1475–1481.

73. Hoshina, A., Kawamoto, T., Sueta, S. I., Mae, S. I., et al. Development of new method to enrich human iPSC-derived renal progenitors using cell surface markers. Sci. Rep. 2018, 8, 6375.

74. Hayashi, R., Ishikawa, Y., Katayama, T., Quantock, A. J., et al. CD200 facilitates the isolation of corneal epithelial cells derived from human pluripotent stem cells. Sci. Rep. 2018, 8, 16550.

75. Santangelo, P., Nitin, N., Bao, G., Nanostructured probes for RNA detection in living cells. Ann. Biomed. Eng. 2006, 34, 39–50.

76. Rhee, W. J., Bao, G., Simultaneous detection of mRNA and protein stem cell markers in live cells. BMC Biotechnol., 2009, 9, 30.

77. Larsson, H. M., Lee, S. T., Roccio, M., Velluto, D., et al. Sorting live stem cells based on Sox2 mRNA expression. PLoS One 2012, 7, e49874.

78. Ban, K., Wile, B., Kim, S., Park, H. J., et al. Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA. Circulation 2013, 128, 1897–1909.

79. Jha, R., Wile, B., Wu, Q., Morris, A. H., et al. Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells. Biomaterials 2015, 50, 176–185.

80. Zhang, J. Z., Termglinchan, V., Shao, N. Y., Itzhaki, I., et al. A human iPSC double-reporter system enables purification of cardiac lineage subpopulations with distinct function and drug response profiles. Cell Stem Cell 2019, 24, 802–811 e805.

81. Yamauchi, K., Li, J., Morikawa, K., Liu, L., et al. Isolation and characterization of ventricular-like cells derived from NKX2-5(eGFP/w) and MLC2v(mCherry/w) double knock-in human pluripotent stem cells. Biochem. Biophys. Res. Commun. 2018, 495, 1278–1284.

82. Li, B., Yang, H., Wang, X., Zhan, Y., et al. Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes, Stem Cell. Res. Ther. 2017, 8, 202.

83. Rojas, S. V., Kensah, G., Rotaermel, A., Baraki, H., et al. Transplantation of purified iPSC-derived cardiomyocytes in myocardial infarction. PLoS One 2017, 12, e0173222.
progenitors by cell sorting for successful transplantation. Stem cell reports 2014, 2, 337–350.

53. Fathi, A., Mirzaei, M., Dolatyar, B., Sharifitabar, M., et al. Discovery of novel cell surface markers for purification of embryonic dopamine progenitors for transplantation in Parkinson's disease animal models. Mol. Cell. Proteomics 2018, 17, 1670–1684.

54. Paik, E. J., O'Neil, A. L., Ng, S. Y., Sun, C., et al. Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture. Sci. Rep. 2018, 8, 804.

55. Samata, B., Doi, D., Nishimura, K., Kikuchi, T., et al. Purification of functional human ES and iPSC-derived midbrain dopaminergic progenitors using LRTM1. Nat Commun 2016, 7, 13097.

56. Sundberg, M., Skottman, H., Suuronen, R., Narkilahti, S., Production and isolation of NG2+ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium. Stem cell res. 2010, 5, 91–103.

57. Djelloul, M., Azevedo, C., Pomeshchik, Y., Hammarberg, A., et al. Reporting on methods to generate and purify rodent and human oligodendrocytes from different sources. Stem Cell Res 2017, 20, 58–66.

58. Alsanie, W. F., Niclis, J. C., Petratos, S., Human embryonic stem cell-derived oligodendrocytes: protocols and perspectives. Stem Cells Dev. 2013, 22, 2459–2476.

59. Yang, L., Soonpaa, M. H., Adler, E. D., Roepke, T. K., et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 2008, 453, 524–528.

60. Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 2011, 8, 228–240.

61. Dubois, N. C., Craft, A. M., Sharma, P., Elliott, D. A., et al. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat. Biotechnol. 2011, 29, 1011–1018.

62. Rust, W., Balakrishnan, T., Zweigerdt, R., Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression. Regen. Med. 2009, 4, 225–237.

63. Schwach, V., Passier, R., Generation and purification of human stem cell-derived cardiomyocytes. Differentiation 2016, 91, 126–138.

64. Tarnawski, L., Xian, X., Monnerat, G., Macaulay, I. C., et al. Integrin based isolation enables purification of murine lineage committed cardiomyocytes. PLoS One 2015, 10, e0135880.

65. Kelly, O. G., Chan, M. Y., Martinson, L. A., Kadoya, K., et al. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat. Biotechnol. 2011, 29, 750–756.

66. Segev, H., Fishman, B., Schulman, R., Itskovitz-Eldor, J., The expression of the class 1 glucose transporter isoforms in human embryonic stem cells, and the potential use of GLUT2 as a marker for pancreatic progenitor enrichment. Stem Cells Dev 2012, 21, 1653–1661.

67. Jiang, W., Sui, X., Zhang, D., Liu, M., et al. CD24: a novel surface marker for PDX1-positive pancreatic progenitors derived from human embryonic stem cells. Stem Cells 2011, 29, 609–617.

68. Wang, P., Rodriguez, R. T., Wang, J., Ghodasara, A., et al. Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell 2011, 8, 335–346.

69. Davenport, C., Diekmann, U., Naujok, O., A quick and efficient method for the purification of endoderm cells generated from human embryonic stem cells. J Vis Exp 2016.

70. Oshima, Y., Suzuki, A., Kawashimo, K., Ishikawa, M., et al. Isolation of mouse pancreatic ductal progenitor cells expressing CD133 and c-Met by flow cytometric cell sorting. Gastroenterology 2007, 132, 720–732.

71. Mallanna, S. K., Cayo, M. A., Twaroski, K., Gundry, R. L., et al. Mapping the cell-surface N-glycoproteome of human hepatocytes reveals markers for selecting a homogeneous population of iPSC-derived hepatocytes. Stem Cell Reports 2016, 7, 543–556.

72. Peters, D. T., Henderson, C. A., Warren, C. R., Friesen, M., et al. Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells. Development 2016, 143, 1475–1481.

73. Hoshina, A., Kawamoto, T., Sueta, S. I., Mae, S. I., et al. Development of new method to enrich human iPSC-derived renal progenitors using cell surface markers. Sci. Rep. 2018, 8, 6375.

74. Hayashi, R., Ishikawa, Y., Katayama, T., Quantock, A. J., et al. CD200 facilitates the isolation of corneal epithelial cells derived from human pluripotent stem cells. Sci. Rep. 2018, 8, 16550.

75. Santangelo, P., Nitin, N., Bao, G., Nanostructured probes for RNA detection in living cells. Ann. Biomed. Eng. 2006, 34, 39–50.

76. Rhee, W. J., Bao, G., Simultaneous detection of mRNA and protein stem cell markers in live cells. BMC Biotechnol., 2009, 9, 30.

77. Larsson, H. M., Lee, S. T., Roccio, M., Velluto, D., et al. Sorting live stem cells based on Sox2 mRNA expression. PLoS One 2012, 7, e49874.

78. Ban, K., Wile, B., Kim, S., Park, H. J., et al. Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA. Circulation 2013, 128, 1897–1909.

79. Jha, R., Wile, B., Wu, Q., Morris, A. H., et al. Molecular beacon-based detection and isolation of working-type cardiomyocytes derived from human pluripotent stem cells. Biomaterials 2015, 50, 176–185.

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<figure><img src="image_7.png" /></figure>

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91. Zhang, R., Mjoseng, H. K., Hoeve, M. A., Bauer, N. G., et al. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells. Nat. Commun. 2013, 4, 1335.

92. Machado, C. B., Kanning, K. C., Kreis, P., Stevenson, D., et al. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons. Development 2014, 141, 784-794.

93. Giobbe, G. G., Zagallo, M., Riello, M., Serena, E., et al. Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells. Biotechnol. Bioeng. 2012, 109, 3119-3132.

94. Hemmi, N., Tohyama, S., Nakajima, K., Kanazawa, H., et al. A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes. Stem Cells Transl Med 2014, 3, 1473-1483.

95. Tohyama, S., Fujita, J., Fujita, C., Yamaguchi, M., et al. Efficient large-scale 2D culture system for human induced pluripotent stem cells and differentiated cardiomyocytes. Stem Cell Reports 2017, 9, 1406-1414.

96. Rathore, A. S., Roadmap for implementation of quality by design (QbD) for biotechnology products. Trends Biotechnol 2009, 27, 546-553.

97. Rathore, A. S., Winkle, H., Quality by design for biopharmaceuticals. Nat Biotechnol 2009, 27, 26-34.

**How to cite this article:** Sart S, Liu C, Zeng EZ, Xu C, Li Y. Downstream bioprocessing of human pluripotent stem cell-derived therapeutics. *Eng Life Sci.* 2022;22:667-680.

https://doi.org/10.1002/elsc.202100042
<figure><img src="image_5.png" /></figure>

84. Wu, J., Hunt, S. D., Xue, H., Liu, Y., et al. Generation and characterization of a MYF5 reporter human iPS cell line using CRISPR/Cas9 mediated homologous recombination. Sci. Rep. 2016, 6, 18759.

85. Wu, J., Matthias, N., Lo, J., Ortiz-Vitali, J. L., et al. A myogenic double-reporter human pluripotent stem cell line allows prospective isolation of skeletal muscle progenitors. Cell Rep., 2018, 25, 1966-1981 e1964.

86. Kim, J., Magli, A., Chan, S. S. K., Oliveira, V. K. P., et al. Expansion and purification are critical for the therapeutic application of pluripotent stem cell-derived myogenic progenitors. Stem Cell Rep. 2017, 9, 12-22.

87. DeRosa, B. A., Belle, K. C., Thomas, B. J., Cukier, H. N., et al. hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells. Mol. Cell. Neurosci. 2015, 68, 244-257.

88. Xia, N., Fang, F., Zhang, P., Cui, J., et al. A knockin reporter allows purification and characterization of mDA neurons from heterogeneous populations. Cell Rep., 2017, 18, 2533-2546.

89. Sart, S., Ma, T., Li, Y., Preconditioning stem cells for in vivo delivery. BioResearch Open Access 2014, 3, 137-149.

90. Nie, Y., Walsh, P., Clarke, D. L., Rowley, J. A., et al. Scalable passaging of adherent human pluripotent stem cells. PLoS One 2014, 9, e88012.

91. Zhang, R., Mjoseng, H. K., Hoeve, M. A., Bauer, N. G., et al. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells. Nat. Commun. 2013, 4, 1335.

92. Machado, C. B., Kanning, K. C., Kreis, P., Stevenson, D., et al. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons. Development 2014, 141, 784-794.

93. Giobbe, G. G., Zagallo, M., Riello, M., Serena, E., et al. Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells. Biotechnol. Bioeng. 2012, 109, 3119-3132.

94. Hemmi, N., Tohyama, S., Nakajima, K., Kanazawa, H., et al. A massive suspension culture system with metabolic purification for human pluripotent stem cell-derived cardiomyocytes. Stem Cells Transl Med 2014, 3, 1473-1483.

95. Tohyama, S., Fujita, J., Fujita, C., Yamaguchi, M., et al. Efficient large-scale 2D culture system for human induced pluripotent stem cells and differentiated cardiomyocytes. Stem Cell Reports 2017, 9, 1406-1414.

96. Rathore, A. S., Roadmap for implementation of quality by design (QbD) for biotechnology products. Trends Biotechnol 2009, 27, 546-553.

97. Rathore, A. S., Winkle, H., Quality by design for biopharmaceuticals. Nat Biotechnol 2009, 27, 26-34.

**How to cite this article:** Sart S, Liu C, Zeng EZ, Xu C, Li Y. Downstream bioprocessing of human pluripotent stem cell-derived therapeutics. *Eng Life Sci.* 2022;22:667-680.

https://doi.org/10.1002/elsc.202100042