BMC Molecular andCell Biology(2024) 25:10
Jiang et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-024-00503-x

Open Access

RESEARCH

Optimization of seeding density of OP9cells to improve hematopoietic differentiation
efficiency
Xin‑xing Jiang1,2,3,4,5,6†, Meng‑yi Song1,2,3,4,5,6†, Qi Li2,3,4,5,6, Yun‑jian Wei2,3,4,5,6, Yuan‑hua Huang1,2,3,4,5,6* andYan‑lin Ma2,3,4,5,6*

Abstract
Background OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem
cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usu‑
ally needs 14–18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system
is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency.
Methods In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h
before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before coculture. Then we compared the hematopoietic differentiation efficiency among them.
Results OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbersof OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were
cultured for 4 days at a total number of 3.1 × ­104 cells/cm2 in a 6-well plate to reach an overgrown state before coculture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these twogroups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × ­104 cells/cm2
in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentia‑
tion efficiency 5 days earlier than the control group.
Conclusion The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total numberof 1.0 × ­106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.
Keywords Embryonic stem cells, Hematopoietic differentiation, OP9 cells, Co-culture†

Xin-xing Jiang and Meng-yi Song contributed equally to this work.

*Correspondence:
Yuan‑hua Huang
13036095796@163.com
Yan‑lin Ma
mayanlinma@163.com
1
The First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, Henan Province, China
2
Hainan Provincial Key Laboratory for Human Reproductive Medicine
and Genetic Research, The First Affiliated Hospital of Hainan Medical
University, Hainan Medical University, Hainan, China
3
Department of Reproductive Medicine, The First Affiliated Hospitalof Hainan Medical University, Hainan Medical University, Hainan, China

4

Hainan Provincial Clinical Research Center for Thalassemia, The
First Affiliated Hospital of Hainan Medical University, Hainan Medical
University, Hainan, China
5
Key Laboratory of Reproductive Health Diseases Research
and Translation (Hainan Medical University, Ministry of Education, Hainan,
China
6
Haikou Key Laboratory for Preservation of Human Genetic Resource,
The First Affiliated Hospital of Hainan Medical University, Hainan Medical
University, Hainan, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

Background
Human pluripotent stem cells (hPSCs), including humanembryonic stem cells (hESCs) and induced pluripotent
stem cells (iPSCs), have an inexhaustible capacity for
self-renewal and differentiation into cells of the threegerm layers. Therefore, PSCs are an attractive source forgenerating hematopoietic stem cells (HSCs) [1, 2], which
are the core of hematopoiesis for their capability of cellproliferation and differentiation into different blood andimmune cells [3, 4]. To date, allo-genetic HSC transplantation (allo-HSCT) is the only definitive cure for many
malignant blood diseases, cancers, and genetic immune
disorders. Primary HSCs are exclusively obtained frombone marrow, mobilized peripheral blood, and cord
blood, however, the major problems are the limitation of
HLA-matched donor sources and the difficulty of obtaining sufficient HSCs for transplantation, and hinder theuse of stem cell therapy in the clinic. For the past several
decades, methods have been developed to direct the differentiation of hPSCs to hematopoietic stem/progenitor cells (HSPCs) in vitro using either formation of theembryonic body (EB) [5], or co-culture with stromal celllines that imitate hematopoietic microenvironment, suchas OP9, S17, and AGM [6–8].
OP9 mouse stromal cell line has been widely used toinduce hPSCs differentiation to HSPCs and multi-lineage
hematopoietic cells [9–11]. One advantage of this co-culture system to induce hESCs differentiation into HSPCs
is that it does not need additional cytokines. However,the disadvantage of this system is its low differentiation
efficiency. The whole co-culture procedure usually needs
14–18 days, including preparing OP9 cells for at least
4 days. Previous studies optimized the OP9 co-culture
system with a focus on adding extrinsic cytokines [10, 12]
and stromal cell gene editing [9].
Traditional differentiation system does not set a precise
density of OP9 cells for co-culture, i.e. overgrown OP9cells were prepared by feeding and prolonged culture ofconfluent OP9 cells, followed by induction of hESCs differentiation into HSPCs for 10 days [6, 13]. We hypothesized that the density of stromal cells in a co-culture
system can influence the efficiency of hematopoietic
differentiation. In the present study, we examined theeffects of different densities of OP9 cells in co-culture
systems and determined the optimal density for efficientdifferentiation. We hope that this optimized system canbe used as a new induction method for in vitro HSPC
differentiation.
Methods
Cell culture

The hESCs line (HN14, 23–30 passages) was obtained
from the Hainan Provincial Key Laboratory for Human

Page 2 of 9

Reproductive Medicine and Genetic Research and maintained on Matrigel-coated plates with hPSCs medium
mTeSR™1 (Stem Cell Technologies, CAN) and passaged
every 3 days at a 1:3 ratio using 0.5 mM EDTA, andchanged fresh medium every day. Cells were maintained
at 37 °C and 5% ­CO2 [14].
OP9 stromal cells (3–15 passages) were purchased fromthe Shanghai Cell Bank of the Chinese Academy of Sciences and cultured on 0.1% gelatinized plates in α-MEM(Gibco, USA) supplemented with 15% fetal bovine
serum (FBS; Gibco, USA) until confluence. When passaged, these cells dissociated with 0.25% trypsin (Gibco)for 1 min at 37 °C. For cryopreservation, OP9 cells weresuspended in 90% FBS added 10% Dimethyl Sulfoxide(Gibco) and stored in a deep freezer at -80 °C overnight,
then transferred to liquid nitrogen.
OP9 cells and HN14 cells were co-cultured in a differentiation medium consisting of α-MEM medium supplemented with 10% defined fetal bovine serum (Hyclone,
USA) and 100 μmol/L monothioglycerol (MTG; Sigma,
USA) [9].
Different co‑culture strategies

OP9 cells were randomly assigned into two groups, experimental and control groups. In the experimental group,
six different seeding numbers of OP9 cells were designed
for each subgroup: 3.1 × ­104cells/cm2, 5.2 × ­104cells/cm2,
7.3 × ­104cells/cm2, 10.4 × ­104cells/cm2, 13.5 × ­104cells/
cm2 and 16.6 × ­104cells/cm2 in 6-well plates. hESCs wereplated on the related stromal cell layer (0.7–1.0 × ­106 cells
in 6-well plate) [9]. These cells were cultured for 1 day
and co-cultured with hESCs. In the control group, OP9cells at a seeding number of 3.1 × ­104cells/cm2 in 6-wellplates were cultured for 4 days to reach a confluent state
followed by co-culture with hESCs. UndifferentiatedhESCs were harvested by treatment with 2 mg/ml dispase enzyme (Gibco, USA) and scraped to maintain thecells in small clumps. The hESCs/OP9 cocultures wereincubated for up to 10 days at 37 °C in normoxic conditions and 5% C
­ O2 with a whole-medium change on day 1
and a half-medium change on days 4, 6, 8, and 10.
Immunofluorescence staining

To determine the identity of cell line OP9, we examined
the immunophenotype of OP9. They were negative for
CD34 and uniformly positive for Calponin [15]. They
were fixed with methanol-acetone (1:1) for 30 min at-20 °C. After washing in phosphate-buffered saline (PBS),
cells were fixed with 0.05% Tween-20 and 10% goat
serum in PBS for 1 h at room temperature, then incubated with primary antibodies against Calponin (Abcam,
USA) and CD34 (Abcam, USA) overnight at 4 °C. Thenthey were washed 3 times and incubated with Donkey

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

Anti-Rabbit IgG (H&L) (Abcam, USA) for an additional
60 min at room temperature. Finally, the cells were incubated with 4′6-diamidino-2-phenylindole (DAPI) for
20 min at room temperature.
Flow cytometry analysis

To analyze the differentiation efficiency of the coculture
system, hESCs/OP9 coculture cells were treated with
0.25% trypsin (Gibco, USA) for 20 min at 37 °C on days
8, 10 and 12. Trypsinization was terminated by adding
a coculture medium. Co-culture cells were harvested bycentrifugation, and washed twice by washing buffer (PBS
supplemented by 5% FBS) [16]. These cells were immediately incubated with APC-human TRA-1–85 (R&D,
USA), PE-Cy5.5 mouse anti-human CD34 (Becton Dickinson Company, USA) for 30 min at 4 °C. After staining,these cells were washed with washing buffer, centrifuged, and resuspended with moderate washing buffer,
followed by flow cytometric analysis (BD FACSAria™ II
sorter (BD Biosciences, USA). Data were analyzed with
FlowJo version 7.6.1 (FlowJo, LLC). For negative control,
APC mouse anti-human CD34 (Becton Dickinson Company, USA) was used to HN14, while OP9 cells labeledanti-CD34 [EP373Y] (Abcam, USA) and used Donkey
Anti-Rabbit IgG (H&L) (Abcam, USA) as the secondary
antibody.
Magnetic sorting for CD34 positive cells

Single-cell suspension from day 10 of hESCs/OP9 cocultures was labeled with CD34 Microbeads Kit (MACS)
as recommended by the manufacturer and went throughan LS separation column attached to a Midi-MACS
Separator to obtain C­ D34+ cells. The purity of isolated
CD34 + cells was always higher than 95% as evaluated bytrypan blue exclusion.
RNA isolation, RT‑PCR, and quantitative RT‑PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA). cDNA was synthesizedusing 1 μg of RNA from each sample with iScript™ cDNA
Synthesis Kit (Promega, USA). RT-PCR cycling conditions were as follows: initial denaturation at 95 °C for
10 min followed by 40 denaturation cycles at 95 °C for
15 s, primer annealing at 60 °C for 60 s, primer extension
phase at 72 °C 15 s and final extension step at 72 °C for
7 min (Agilent Stratagene Mx3000P, Agilent Technologies, USA). GAPDH was used as an internal control. All
experiments were performed three times. Sequences ofprimers used in this study are shown in Table 1.
Colony‑forming assay

Sorted ­CD34+ cells were plated at a density of 2.5 × ­104
cells/dish and suspended in 1 ml methylcellulose medium

Page 3 of 9

Table 1 Primer sequences for RT-PCR
GeneForward (5’ to 3’)

Reverse (5’ to 3’)

GAPDH CGA​GAT​CCC​TCC​AAA​ATC​AA

TGT​GGT​CAT​GAG​TCC​T TC​CA

CD31

CCA​AGG​TGG​GAT​CGT​GAG​G

TCG​GAA​GGA​TAA​AAC​GCG​GTC​

CD34

CTA​CAA​CAC​C TA​GTA​CCC​
TTGGA​

GGT​GAA​CAC​TGT​GCT​GAT​TACA​

CD43

TCA​GCC​C TA​CCT​CCC​TCA​ACTAT​GGT​TCC​ACC​TGT​CAC​GGT​(MethoCult H4434, Stem Cell Technologies) in 35 mm
low adherent dishes at 37 °C in a humidified atmosphereof 5% ­CO2 for 14–16 days. All clonogenic progenitor
assays were performed in duplicate. Colonies of morethan 50 cells were scored as positive clones and defined
as macrophage (M), granulocyte and macrophage (GM),
granulocyte (G), erythroid (E) according to their colonymorphology.
Statistical analysis

Data were analyzed with Prism 5 software (GraphPad,
USA), and are presented shown as the mean ± SD. Significant differences were considered at the P < 0.05 level.

Results
Identification of OP9 cells and differentiation of hESCs

The OP9 cells were bipolar fibroblast-like in morphology. They were morphologically homogeneous populations. Immunofluorescence staining was used to assess
the molecular characteristics of the OP9 (Fig. 1A),
the OP9 cells were negative for CD34 and expresseda marker of Calponin. As shown in Fig. 1D, OP9 cells
were cultured for 1 day with six different seeding numbers in 6-well plates: a to f were the morphology of different seeding numbers,3.1 × ­104cells/cm2,5.2 × ­104cells/
cm2,7.3 × ­104cells/cm2,10.4 × ­104cells/cm2,13.5 × ­104cells/
cm2and 16.6 × ­104cells/cm2, respectively. OP9 cells plated
at the numbers of 1.0–1.3 × ­106 cells reached nearly 100%confluency after 1 day of culture (d, e). When the seeding
number of OP9 cells could reach 16.6 × ­104cells/cm2, thecultures reached confluence. In the control group, overgrown OP9 cultures were prepared for 4 days (Fig. 1C).
HN14 cells were plated on the stromal cell layer when
they reached 80% confluency. During the differentiation process, the morphology of cells started to change
on days 8, 10, and 12 and hESCs started differentiating through the morphologic observation (Fig. 1B). As
shown in Fig. 2, HN14 cells seeded on six different numbers of OP9 cells, 3.1 × ­104 cells, 5.2 × ­104 cells, 7.3 × ­104
cells, 10.4 × ­104 cells, 13.5 × ­104 cells, and 16.6 × ­104 cells
per square centimeter, respectively, on days 8, 10 or 12(bar = 100 μm).

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

Fig. 1 Identification and morphology of OP9 cells and hESCs
before co-culture. A Immunophenotype of OP9 cells.
Immunofluorescence staining of Calponin and CD34 in the OP9.
Original magnification: × 40. B The morphology of hESCs (HN14)
before co-culture (bar = 100 μm). hESCs monolayers at 80%
confluence and co-cultured with OP9 cells. C The morphology of OP9cells in the control group. OP9 cells reached confluence for culturing
for 4 days at a seeding number of 3.1 × ­104 cells/cm2 in 6-well plates(bar = 100 μm). D The morphology of OP9 cells in the experimentalgroup. OP9 cells were cultured for 1 day with six different seeding
numbers in 6-well plates: a to f show the morphology of different
seeding densities, i.e. 3.1 × ­104 cells, 5.2 × ­104 cells, 7.3 × ­104 cells,
10.4 × ­104 cells, 13.5 × ­104 cells, and 16.6 × ­104 cells per squarecentimeter, respectively (bar = 100 μm). When the seeding numberof OP9 cells could reach 16.6 × ­104cells/cm2, the cultures reachedconfluence

Effects of seeding density on the expression of CD34,
CD43, and CD31

Using flow cytometry analysis, we could easily distinguish human cells from OP9 because of APC-human
TRA-1–85. CD34 is an important surface marker for
HSPCs and can define HSPCs in the human hESCs/OP9
co-culture system [15]. CD34 was not expressed in original cells including HN14 and OP9 cells (Fig. 3A). During
differentiation, we harvested co-culture cells on days 8,
10 and 12, and analyzed the expression of CD34 by flow
cytometry. As shown in Fig. 3C, a to f were the six different seeding numbers of OP9 cells from 3.1 × ­104 cells
to 16.6 × ­104 cells per square centimeter in 6-well plates.
With the increasing density of OP9 cells, the efficiency ofhematopoietic differentiation was improved from 2.47%
to 6.27%. The group of seeding number of 10.4 × ­104cells
resulted in 10% of C
­ D34+ cells or even more on day 10.
Except for the seeding number of 3 × ­105cells in a 6-wellplate, all other groups had a peak of ­CD34+ cells on day
10 during hematopoietic differentiation. However, thecontrol group had a peak of CD34 + cells on day 12 orlater (Fig. 3B). These findings suggested that optimizing
the seeding density of OP9 cells improves hESCs hematopoietic differentiation. Consistent with the results ofPage 4 of 9

flow cytometry, the expression of CD34 mRNA between
10,4 × ­104cells, 13.5 × ­104 cells, and 16.6 × ­104 cells/cm2
groups was comparable (Fig. 3D).
We also investigated the expression of CD31 and CD43
in these cells on day 10 (Fig. 3C) by qRT-PCR. CD43 andCD31 were also markers of HSPCs [3]. When the seeding number was up to 16.6 × ­104 cells per square centimeter in a 6-well plate, the expression of CD43 wasincreasing. We also found that when the seeding number was 10.4 × ­104 cells per square centimeter in a 6-wellplate, CD31 expression increased more obviously thanother densities. Likewise, the expression level of CD43
and CD31 reached a peak on day 10 in the experimentalgroup.
No effect of seeding density on the differentiationof HSPCs

The purpose of this study was to optimize the co-culture
system. We compared the expression of CD34, CD31,
and CD43 between the experimental group on day 10
and the control group on day 12. The expression of CD34,
CD31 and CD43 had no significant difference (Fig. 4A).
We enriched CD34 + cells with magnetic cell sorting(Fig. 4B and C) and promoted them to differentiate into
different CFUs, such as macrophage (M), granulocyte
and macrophage (GM), granulocyte (G), erythroid (E).
Consistent with the observations from flow cytometry
and RT-qPCR, the proficiency of further differentiation
had no difference between the two groups when theseeding density reached 5.0 × ­105cells/ml.

Discussion
Stromal cells are one of the most important cell types
in bone marrow that contain the hematopoietic niche,and hematopoiesis is regulated by microenvironment or
niche [17, 18]. Some studies attempted to derive robustly
induced HPCs from PSCs in vitro by simulating hematopoietic niches [18–20], and others tried to improve
techniques to mimic the hematopoietic niche. The objective of these studies was to improve HSC differentiation
to a large scale so that it may be used in regenerative
medicine applications.
The co-culture technique was used for many applications, and the efficiency of differentiation was closely
related to the condition of the stromal cells. Stromal cells
producing M-CSF induce the differentiation of embryonic stem cells (ESC) down the monocyte-macrophagelineage [20, 21]. Nevertheless, OP9 stromal cells lacking
M-CSF promote diferentiation down other haematopoietic lineages [6, 22, 23].
OP9 cells are one of the stromal cells that have been
widely used because they provide more efficient support of hematopoiesis than other stromal cell lines

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

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Fig. 2 The morphology of ES cells on OP9 stromal cells during differentiation on days 8, 10 and 12. (A) a to f show the morphological changesof HN14 cells on six different seeding numbers of OP9 cells, i.e. 3.1 × ­104 cells, 5.2 × ­104 cells, 7.3 × ­104 cells, 10.4 × ­104 cells, 13.5 × ­104 cells,and 16.6 × ­104 cells/cm2, respectively, on days 8, 10 or 12 (bar = 100 μm). B The morphologies of differentiated HN14 colonies during co-culture
on days 8, 10 and 12 in the control group (bar = 100 μm). In all groups, the presence of rounded cells at the edge of the colonies

probably due to that OP9 could release paracrine mediated signaling [24, 25]. In previous studies, hPSCs
seeded onto overconfluent OP9 stroma, after 12 daysof co-culture, with OP9 cells, the percentage of CD34
positive cells reached 10.3 ± 1.8%, but did not define theseed density and culture time before co-culture [26]. As
the efficiency of co-culture was not stable, most of thestudies focused on modification of gene expression, thefunction of OP9 stromal cells, or add hematopoieticrelated cytokines to improve differentiation efficiency,

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

Page 6 of 9

Fig. 3 Determination of differentiation efficiency in the hESCs/OP9 co-culture system. A The expression of CD34 in undifferentiated humanembryonic stem cells (HN14) and OP9 cells, separately. B Percentage of ­CD34+ cells by flow cytometry (x-axis) in the control group. The peakof hematopoietic differentiation on D12. C Percentage of ­CD34+ cells by flow cytometry (x-axis) in the experimental group. a to f show six
different seeding numbers in 6-well plates, 3.1 × ­104 cells, 5.2 × ­104 cells, 7.3 × ­104 cells, 10.4 × ­104 cells, 13.5 × ­104 cells, and 16.6 × ­104 cells/cm2,
respectively. Single-cell suspension from hESCs/OP9 co-culture obtained at the indicated time points was labeled with CD34 (x-axis). The number
indicates the percentage of positive cells in the corresponding group. Except for (a) group, the peak of hematopoietic differentiation on D10.
D Evaluation of the expression of hematopoietic-related genes on day 10 in the experimental group. RNA was extracted, and gene expression
was quantified by qRT-PCR. Statistical results of phase-specific genes are shown as the mean ± SD, n = 3. ## P < 0.01 and # P < 0.05 comparedwith the 16.6 × ­104 cells/cm2 group. ▲▲ P < 0.01 and ▲ P < 0.05 compared with the 13.5 × ­104 cells/cm2 group. ★★ P < 0.01 and ★ P < 0.05
compared with the 10.4 × ­104 cells/cm2 groupprevious studies showed that over-expressed transcription factor Lhx2 onto OP9 cells [9] or modified extrinsic factors directly on OP9 cells [27] enhanced hPSCs

hematopoietic differentiation. With an in-depth understanding of hematopoietic differentiation, OP9 cells
were modified with Notch ligand delta-like 1 to develop

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10

Page 7 of 9

Fig. 4 Improving the initial seeding number of OP9 cells shortened the whole time of co-culture system without changing the efficiencyof hematopoietic differentiation. A Comparison of the expression of hematopoietic-related genes during differentiation between the experimental
subgroup (10.4 × ­104 cells, 13.5 × ­104 cells, and 16.6 × ­104 cells/cm2 in a six-well plate) and the control group. Data are presented as means ± SD
(n = 3). These results were consistent with flow cytometry. B Cell distribution in CFU from sorted CD34 positive cells for culturing 14–16 daysin a Semi-solid medium. E, erythroid; G, granulocyte; GM, granulocyte/macrophage; M, macrophage. C Morphology of different colony-forming unit
types, including M, GM, G and E. Scale bar = 100 mm

T lymphocytes [28] and NK cells [29]. Importantly, OP9cells secreted factors and signaling molecules to induce
hematopoietic differentiation [30, 31]. One study usedsingle-cell q-PCR to track the dynamic gene expressionof OP9-hESCs co-culture system at different stages andtransiently over-expressed specific transcription factors

Jiang et al. BMC Molecular and Cell Biology(2024) 25:10(TFs) at different stages [32] and showed that overexpression of these TFs mainly promoted differentiation at the initial stage. Another recent study used 3D
cell culture by co-culture of OP9 cells, and provided aminimal, scalable, biomimetic in vitro model of hematopoietic differentiation in the absence of exogenous
cytokines [33]. Although OP9 stromal cells are universally used in hematopoietic differentiation. The efficiency of HSPC generation is related to the density ofOP9 cells, however, no study explored the association
between the density of OP9 cells with hematopoietic
differentiation. We suggest that the longer OP9 cells
were cultured, the more aging OP9 cells would become,and aging OP9 cells are prone to fat degeneration andinsufficiency of secreting cytokines.
In the present study, to define the density of OP9cells, we established an optimized OP9 co-culture system without adding any recombinant cytokines (Fig. 5).
We focused on the seeding density of OP9 cells in the
co-culture system. A seeding number of 16.6 × ­104
cells per square centimeter in a 6-well plate of OP9cells was able to reduce culture time without significantly affecting the differentiation efficiency compared
to previous study [26]. For example, the initial seeding
density of OP9 cells at 10.4 × ­104 cells/cm2 in a 6-wellplate had 10% percent of C
­ D34+ cells at day 10, compared with day 12 in the control group. Increasing thedensity of OP9 cells further increased the percentageof ­C D34+ but showed no significant difference between
the control and experimental groups. In other words,
we reduced the differentiation time by 2 days with thesame differentiation of efficiency. In addition, in our
optimized OP9 co-culture system, HSPCs successfully
differentiated into different CFUs (colony-forming
units) with comparable efficiency, which meant that
this system had the potential to produce multipotent
hematopoietic progenitors. Thus, we have optimized
the co-culture system to reduce the differentiation
time for at least 5 days without changing differentiation
efficiency.

Page 8 of 9

Conclusions
Our optimized approach is a novel, faster, and lowcost method to induce hematopoietic differentiationin vitro. However, the underlying mechanisms remain
unclear. Future studies are needed to identify the possible factors or signaling molecules secreted by OP9cells at different seeding densities and determine whichis responsible for the improved differentiation observedin the present study. In the future, we will test related
factors to induce differentiation and further optimize
the differentiation system in the absence of OP9 cells.
Abbreviations
hESC	Human embryonic stem cells
HSPC	Hematopoietic stem/progenitor cell
HPSC	Human pluripotent stem cells
iPSC	Induced pluripotent stem cellEB	Embryonic body
TF	Transcription factors
CFU	Colony-forming units
Acknowledgements
We thank all staff in the Department of Reproductive Medicine, the First Affili‑
ated Hospital of Hainan Medical University (especially Dr. Fei Sun) for their help
and support.
Authors’ contributions
J.X., M.Y., L.Q. and H.Y. initiated and designed the project. J.X., M.Y. and L.Q.
wrote the manuscript. W.Y. and J.X. performed most experiments and analyzed
the result data. H.Y., and M.Y. supervise the designed structure of the manu‑
script, review and help revise this manuscript.
Funding
This work was supported in part by grants from the Major Science and Technol‑
ogy Program of Hainan Province (ZDK2021037), and Hainan Province Scienceand Technology Special Fund (ZDYF2020117, ZDYF2022SHFZ312), Hainan Prov‑
ince Science and Technology Project (LCXY202102, LCYX202203, LCYX202301)
the project supported by Hainan Province Clinical Medical Center, the specificresearch fund of The Innovation Platform for Academicians of Hainan Province.
Availability of data and materials
No datasets were generated or analysed during the current study.

Declarations
Ethics approval and consent to participate
The study was approved by the Research Ethics Committee of the First Affili‑
ated Hospital of Hainan Medical University.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 29 November 2023 Accepted: 1 March 2024

Fig. 5 Schematic diagram showing two different differentiation
systems. As shown in the schematic diagram, the optimized method
could shorten the whole differentiation procedure by nearly 5 days

References
1. Palacios R, Golunski E, Samaridis J. In vitro generation of hematopoietic
stem cells from an embryonic stem cell line. Proc Natl Acad Sci U S A.
1995;92:7530–4.

Jiang et al. BMC Molecular and Cell Biology

2.
3.
4.
5.
6.

7.
8.

9.

10.

11.

12.

13.
14.
15.
16.
17.
18.
19.
20.

21.
22.
23.

24.

(2024) 25:10

Niwa A, Umeda K, Chang H, Saito M, Okita K, et al. Orderly hematopoietic
development of induced pluripotent stem cells via Flk-1(+) hemoangio‑
genic progenitors. J Cell Physiol. 2009;221:367–77.
Morrison SJUN, Weissman IL. The biology of hematopoietic stem cells.
Annu Rev Cell Dev Biol. 1995;11:35–71.
Weissman IL. Stem cells: units of development, units of regeneration, andunits in evolution. Cell. 2000;100:157–68.
Song B, Fan Y, He W, Zhu D, Niu X, et al. Improved hematopoietic differ‑entiation efficiency of gene-corrected beta-thalassemia induced pluripo‑
tent stem cells by CRISPR/Cas9 system. Stem Cells Dev. 2015;24:1053–65.
Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem
cell-derived CD34+ cells: efficient production in the coculture with OP9stromal cells and analysis of lymphohematopoietic potential. Blood.
2005;105:617–26.
Tian XWP, Morris JK, Linehan JL, Kaufman DS. Hematopoietic engraftmentof human embryonic stem cell derived cells is regulated by recipient
innate immunity. Stem Cells. 2006;24:1370–80.
Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, et al.
Efficient hematopoietic differentiation of human embryonic stem cells
on stromal cells derived from hematopoietic niches. Cell Stem Cell.
2008;3:85–98.
Chen XZQ, Li C, Geng Y, Huang K, Zhang J, Wang X, Yang J, Wang T, Xia
C, Liu X, Meng M, Yang D, Zheng Y, Du J, Zhang X, Chen J, Pan G, Wang J.
OP9-Lhx2 stromal cells facilitate derivation of hematopoietic progenitors
both in vitro and in vivo. Stem Cell Reseach. 2015;15:395–402.
Kennedy M, Awong G, Sturgeon CM, Ditadi A, LaMotte-Mohs R, et al. T
lymphocyte potential marks the emergence of definitive hematopoietic
progenitors in human pluripotent stem cell differentiation cultures. Cell
Rep. 2012;2:1722–35.
Yang CTFA, Goh PA, Pagnamenta A, Mettananda S, Taylor J, Knight
S, Nathwani A, Roberts DJ, Watt SM, Carpenter L. Human induced
pluripotent stem cell derived erythroblasts can undergo definitive
erythropoiesis and co-express gamma and beta globins. Br J Haematol.
2014;166:435–48.
Choi KD, Vodyanik MA, Togarrati PP, Suknuntha K, Kumar A, et al. Identifi‑
cation of the hemogenic endothelial progenitor and its direct precursor
in human pluripotent stem cell differentiation cultures. Cell Rep.
2012;2:553–67.
Vodyanik MA, Thomson JA, Slukvin II. Leukosialin (CD43) defines
hematopoietic progenitors in human embryonic stem cell differentiation
cultures. Blood. 2006;108:2095–105.
Huang K, Zhu Y, Ma Y, et al. BMI1 enables interspecies chimerism withhuman pluripotent stem cells. Nat Commun. 2018;9(1):4649.
Gao J, Yan XL, et al. Characterization of OP9 as authentic mesenchymal
stem cell line. J Genet Genomics. 2010;37(7):475–82.
Fathi E, Valipour B, et al. Targeting the proliferation inhibition of chronic
myeloid leukemia cells by bone marrow derived-mesenchymal stem cells
via ERK pathway as a therapeutic strategy. Acta Med Iran. 2020;58(5):199.
Chadwick K, Wang L, Li L, Menendez P, Murdoch B, et al. Cytokines and
BMP-4 promote hematopoietic differentiation of human embryonic stem
cells. Blood. 2003;102:906–15.
Morrison SJSD. The bone marrow niche for haematopoietic stem cells.
Nature. 2014;505:327–34.
Crane GM, Jeffery E, Morrison SJ. Adult haematopoietic stem cell niches.
Nat Rev Immunol. 2017;17:573–90.
Klimchenko O, Mori M, Distefano A, Langlois T, Larbret F, et al. A com‑
mon bipotent progenitor generates the erythroid and megakaryocyte
lineages in embryonic stem cell-derived primitive hematopoiesis. Blood.
2009;114:1506–17.
Nakano T, Kodama H, Honjo T. Generation of lymphohematopoietic cells
from embryonic stem cells in culture. Science. 1994;265:1098–101.
Dias J, et al. Generation of red blood cells from human induced pluripo‑
tent stem cells. Stem Cells Dev. 2011;20:1639–47.
Trakarnsanga K, et al. Qualitative and quantitative comparison of theproteome of erythroid cells diferentiated from human iPSCs and adulterythroid cells by multiplex TMT labelling and nanoLC-MS/MS. PLoS ONE.
2014;9:e100874.
McKinney-Freeman SL, Naveiras O. et al. Isolation of hematopoietic stem
cells from mouse embryonic stem cells. Curr Protoc Stem Cell Biol Chap‑
ter 1: Unit 1F. 2008;3. https://​doi.​org/​10.​1002/​97804​70151​808.​sc01f​03s4.

Page 9 of 925. Ji J, Vijayaragavan K, et al. OP9 stroma augments survival of hematopoi‑
etic precursors and progenitors during hematopoietic differentiationfrom human embryonic stem cells. Stem Cells. 2008;26(10):2485–95.
26. French A, Yang CT, Taylor S, Watt SM, Carpenter L. Human induced
pluripotent stem cell-derived B lymphocytes express sIgM and can begenerated via a hemogenic endothelium intermediate. Stem Cells Dev.
2015;24(9):1082–95.
27. Ichii M, Frank MB, Iozzo RV, Kincade PW. The canonical Wnt pathway
shapes niches supportive of hematopoietic stem/progenitor cells. Blood.
2012;119:1683–92.
28. Kaneko S. In vitro generation of antigen-specific T cells from induced
pluripotent stem cells of antigen-specific T cell origin. Methods Mol Bio.
2016;1393:67.
29. Kyoizumi S, Kubo Y, Kajimura J, Yoshida K, Imai K, Hayashi T, et al. Ageassociated changes in the differentiation potentials of human circulat‑
ing hematopoietic progenitors to T- or NK-lineage cells. J Immunol.
2013;190(12):6164.
30. Figueiredo LM, Costa EB, Orellana MD, Picanco-Castro V, Covas DT. OP9
Stromal Cells Proteins Involved in Hematoendothelial Differentiation from
Human Embryonic Stem Cells. Cell Reprogram. 2015;17:338–46.
31. Trakarnsanga K, Wilson MC, Heesom KJ, Andrienko TN, Srisawat C, Frayne
J. Secretory factors from OP9 stromal cells delay differentiation andincrease the expansion potential of adult erythroid cells in vitro. Sci Rep.
2018;8:1983.
32. Chen H, Jiang M, Xiao L, Huang H. Single-cell qPCR facilitates the optimi‑
zation of hematopoietic differentiation in hPSCs/OP9 coculture system.
Braz J Med Biol Res. 2018;51:e7183.
33. Tavakol DN, Tratwal J, Bonini F, Genta M, Campos V, et al. Injectable, scal‑
able 3D tissue-engineered model of marrow hematopoiesis. Biomaterials.
2020;232:119665.

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