Stem Cells and Differentiation
Rebecca Quelch1 and Stefan Przyborski1,2 1Department of Biosciences, Durham University, South Road, Durham, UK. 2ReproCELL Europe Ltd, NET Park Incubator, Sedgefield, UK. Email: s.a.przyborski@durham.ac.uk
Stem cells are defined by their ability to self-renew and propensity to differentiate into functional cell types. Pluripotent Stem Cells (PSCs) differentiate into cells of all three germ layers (endoderm, ectoderm and mesoderm), whereas multipotent
stem cells are more limited in their differentiation potential. The unique abilities of these cell types make them attractive tools for a wide range of applications, from regenerative medicine to drug toxicity screening. There are a large number ofprotocols for the maintenance of this pluripotent state, as well as for the subsequent directed differentiation of these cells in vitro to form specialized cell types.

Pluripotent Stem Cells (PSCs) In Vitro

Monolayer Differentiation of Pluripotent Stem Cells

The main pluripotent cell types used in vitro have been isolated over a number of decades.

Initial models of differentiation were based upon conventional monolayer cell culture, and facilitated identification of the key biochemical
pathways controlling spatial patterning and development: canonical WNT, Hedgehog, TGF and Retinoic Acid. Using this method many key
modulators governing these pathways were also revealed, providing well characterized and controllable methods of differentiation. There are anumber of known disadvantages of monolayer systems, the main one of which, a lack of biomimetic extracellular architecture, is thought to be
the main contributor to the extremely low yields of differentiation plaguing regenerative medicine. There are, however, a number of establishedand emerging alternative techniques that researchers are now turning to.

1967 & 1975 Isolation of Mouse and Human Embryonal Carcinoma (EC) Cells [1 & 2]
Enzymatic
dissociation

Ascitesteratoma

Single cellisolation andcultivation

Products Available
From Tocris
CHIR 99021,
Dorsomorphin
Mesoderm

LDN 193189,
SB 431542

IDE1
Endoderm

Ectoderm

Embryoid body

Current and Emerging Trends in SC Differentiation

1981 Mouse Embryonic Stem (mES) Cell Isolation [3]Mouse blastocystSeparatecolonies

Inner cellmass

3D Differentiation of PSCs: Embryoid Bodies

Culture on mitomycin C-inactivated
STO ﬁbroblast feeder layer in vitro

4 daysThree dimensional (3D) differentiation of PSCs has been utilized since the 1980s, throughcell aggregation in the absence of an adhesive culture substrate, which exploits the known
PSC dependency upon cell-cell contact (e.g. E-cadherin) signaling. Aggregate formation
can be utilized for the expansion and differentiation of pluripotent cells. In the presence offactors to maintain self-renewal, such as LIF (for mouse ESCs), cells aggregate but do notdifferentiate, an attractive prospect for scaling up cell numbers for regenerative medicine.
In the absence of factors used to maintain pluripotency, aggregate, or more specifically,
embryoid body (EB) formation occurs resulting in spontaneous cell differentiation. EBs
recapitulate developmental processes in a more relevant way than two dimensional(2D) culture and have certain other advantages including scalability. EBs can be formed
in a number of ways, including hanging drop culture systems or microwell plates,
providing good consistency in aggregate size and so limiting variability within a culture.
Disadvantages however include a rather haphazard differentiation profile when notmodulated exogenously, and often the formation of a necrotic core, limiting the time spanof differentiation studies. Differentiation can be directed generally towards each germlayer, by use of a combination of small molecules and further structural support such as
Matrigel® or modulated very precisely to form organoids that represent complex in vitro
aggregate structures that resemble specific organ types.

Can be maintained
indeﬁnitely in vitro

1998 Human Embryonic Stem (hES) Cell Isolation [4]2007 Induced Pluripotent Stem (iPS) Cell Isolation [5]Oct3/4 Klf4
Sox2 C-Myc

iPS

Somatic cellisolation

Adult human

Human skinﬁbroblasts

Yamanaka
factor treatment

Induced pluripotent
stem cells

Since EB formation is controllable by small molecule modulators of key pathways governing differentiation, a similar process could
be used to form primitive organs in vitro. These structures can be formed from differentiated cell types, more commonly termed
spheroids, or using stem cells, including iPSCs, termed organoids. Spheroids are formed by much less complex methods thanorganoids, usually only capitalizing on the propensity of all cells to aggregate in low adherence conditions, and their functionality
reﬂects this. Organoids are generally much more complex in nature than spheroids, mimicking more closely the in vivo condition of a
selected cell type. While this research theme is emerging rapidly, and is proving to have a vast range of applications, there are alreadya number of models of different organs that impressively recapitulate in vivo morphology and physiology.

Somatic cellPSC
Differentiation

Lineage speciﬁcation

Reprogramming
Adult Stem Cells/Progenitors

LIF or Y-27632

Differentiation
Aggregation in3D culture

Adult Stem Cells (ASCs)
It is thought that most organs contain resident adult (or somatic) stem cell populations, i.e. groups of cells that are able toself-renew and differentiate into a limited number of cell types to repopulate an organ for maintenance of functionality
throughout normal cell turnover or upon challenge/injury. Mesenchymal stem cells (MSCs) are a very well characterized
somatic stem cell type, due to their ease of isolation from connective tissue and relatively large capacity for differentiation.
Recently, the receptor LGR5 has gained provenance as a marker for a range of more speciﬁc organ adult cell types, such as
in the intestine and liver. Such markers are important in the development of organoid technology

SC-derived Organoids:
Promising Tools for a Range of Applications

mESCs

Adipocyte

MyocyteOsteogenesis

Osteocyte

Chondrogenesis

ChondrocyteFibrogenesis

Fibroblast

More recently focus has shifted further towards the composition of the growth substrate itself, including substratecoatings (eg. tailored integrins) and substrate topography (hydrogels and other scaffolds) for both the maintenance anddifferentiation of PSCs.

Embryoid body

The major alternative mode of 3D differentiation is the use of scaffolds to recapitulate
cellular microenvironments. These are produced using naturally occurring or synthetic
materials taking a wide variety of physical characteristics. Synthetic methods, such as
electrospinning and polyHIPE formation are popular options for cell differentiation,lending themselves well to engineering tissues such as bone, using an often increased
substrate stiffness to drive differentiation mechanically. Decellularized extracellular matrix(ECM) scaffolds have shown success when used with MSCs for cartilage engineering, withpotential in joint repair. However, methods such as this rely heavily on the maintenanceof the native topography of the ECM for effective repopulation, and removal of all
cellular material to avoid immune rejection. The final major type of scaffold consists ofhydrogels; used to encapsulate a wide range of cell types, including PSCs, progenitorsand differentiated populations. This technology is highly tuneable, in terms of viscosity,porosity and more.

b

Cells allowed to aggregate in
low adherence vessels

Application of specialized cues to drive differentiation (for example;
exogenous small molecules in growth medium for signaling pathway
modulation, extracellular scaffolding components (e.g. hydrogels) orintroducing ﬂuid ﬂow)
Organoids

Advantages/disadvantages:
Advantages include:• Low cost• Ease of culture
Disadvantages include:• Lack of complexity• Potential necrotic coreformation

Cerebral organoid

Intestinal organoid

Gastric organoid

Applications:• Drug screening• Developmental study• Personalized medicine• Modeling of disease states

Future Perspectives
As organoid technology is a rapidly developing field, there are high hopes for its use in a range of applications. In addition toforming complex tissues, organoid formation is also being used to model embryogenesis itself through similarly formed structures
known as gastruloids. By modulating the activation of developmental biochemical pathways, coupled with the biophysical cellular
microenvironment, it is possible to emulate the early events governing embryo development. It is hoped this area of research will
provide never previously achievable insight into such events.
The formation of complex tissue-like systems in vitro provides an invaluable tool to decipher the intricate mechanisms that control
development among multiple cell types. This may then in turn lead to an increased understanding applicable to the isolation of moredifficult differentiated cell populations.

Examples of 3D Culture Scaffoldsa Biomimetic fiber-hydrogel scaffold
b Nanogroove/ridge array

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Proliferation and Cell Viability
A 769662, A 83-01, BIO, CH 223191,
Chroman 1, Emricasan, Go 6983, IWP 2,
LY 294002, Prostaglandin E2 , PD 173074,
PD 98059, SB 202190, SB 203580,
SB 216763, SB 431542, trans-ISRIB,
Troglitazone, U0126, Y-27632
Stem Cell Signaling
AMD 3100, Cyclopamine, IWP 2,
Mitomycin C, XAV 939

Stem Cell Products Available
From Other Bio-Techne Brands

Maintenance of Stem Cell Phenotype In Vitro
The maintenance of stem cell self-renewal and an undifferentiated status is often overlooked. However stem cells occupy
a continuum of developmental states (e.g. “naïve” vs. “primed” pluripotency) that can, in some cases, be defined by theculture environment. Methods of maintaining pluripotency were initially focused around growth on “feeder” layers offibroblasts. The spotlight subsequently shifted to specialized growth medium formulations and the use of qualified serumand small molecules. LIF for mESCs and bFGF for hESCs, are most commonly used to maintain pluripotency, but there are arange of other compounds, including Y-27632, A 83-01 or SB 431542, which can be used to maintain cell phenotype.

Mesenchymal Stem Cells
AICAR, Dexamethasone, Purmorphamine,
SP 600125 , Zebularine

Differentiation

Spheroida

Cardiomyocyte
Dorsomorphin, 1-EBIO, ISX 9, IWP 4,
Wnt-C59

Storage
DMSO Cell Cryopreserve Grade, Y-27632

Alternative 3D Differentiation Approaches

Myogenesis

Differentiation
Neural Stem Cells
DAPT, Dibutyryl-cAMP, Forskolin,Fluoxetine, IBMX, LDN 193189,Metformin, Retinoic Acid,
SAG dihydrochloride, SU 5402

Proliferation

Mesenchymal stem cell

Adipogenesis

Reprogramming(±)-Bay K 8644, BIX 01294,
CHIR 99021, DBZ, 3 -Deazaneplanocin A,Kenpaullone, L-Ascorbic acid,
PD 0325901, RepSox, (S)-(+)Dimethindene, SMER 28, Thiazovivin,
Trichostatin A, Valproic acid, sodium salt

Another major area of interest is that of personalized medicine. Primary cells derived from individuals have also been shown toform these structures, opening avenues into targeted therapies tailored to individuals. This is of great interest to pharmaceuticalcompanies looking at developing high throughput techniques, to provide better predictive validity of candidate drug screens.

Learn more | tocris.com

Cell Culture and Differentiation
Cultrex™ Stem Cell-Qualified BME – ECM
scaffolding for stem cells
Cultrex™ BME, Type 2 – ECM scaffolding fororganoid growth
N21-MAX – Media supplement to support
stem cell and organoid healthN-2 MAX – Media supplement to support stem
cell and organoid health
Verifying Pluripotency and Stemness
Human Pluripotent Stem Cell Functional
Identification Kit
GloLIVE™ Human Pluripotent Stem Cell Live
Cell Imaging Kit
Growth Factors and Proteins
Recombinant Human/Mouse/Rat/Canine/Equine BDNF Protein
rhFGF basic (146 aa) Protein
rhGDNF Protein
rhFGF basic (146 aa) Protein
rhLIF Protein
Abbreviations
EC, Embryonal Carcinoma; ECM, Extracellular Matrix; ES, Embryonic
Stem; iPS, Induced Pluripotent Stem; AS, Adult Stem; EB, Embryoid
Body; MSC, Mesenchymal Stem Cell; 2D, two dimensional; 3D, threedimensional
References:
1 Finch, BW and Ephrussi, B (1967) Proc.Natl.Acad.Sci. U.S.A. 57 615
2 Fogh, J and Tempe, G (1975) New human tumor cell lines. In HumanTumor Cells In Vitro. J. Fogh, (ed.) 115–159, Plenum, New York,
3 Evans, HJ and Kaufman, MH (1981) Nature 292 154
4 Thomson, J et al (1998)
5 Takahashi, K et al (2007) Cell 131 861
Recommended Reviews
Dutta, D et al (2017) Trends Mol. Med. 23 393
Clarke, K et al (2015) Tocris scientific review series: Using small molecules
to control stem cell growth and differentiation.
NOTE: This poster conveys a general overview and should beconsidered neither comprehensive nor definitive. The details of
this information are understood to be subject to interpretation.

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