The role of small molecules in stem cell biology - mini-review

Role of small molecules in stem cell biologyOverview

Stem cells have many potential applications in medicine  - ranging from their inclusion in disease modeling and drug discovery to cell transplantation and regenerative therapies. Challenges in the field include developing methods to control stem cell differentiation, allogeneic rejection and limited cell availability. A growing range of small molecules has been identified that can be used both in vitro and in vivo as tools to expand stem cells, direct their differentiation, or reprogram somatic cells to a more naive state. These molecules can also provide useful information regarding the signaling and epigenetic mechanisms that regulate stem cell biology. This mini review highlights the applications and advantages of small molecules in key areas of stem cell biology including reprogramming, self renewal, differentiation and proliferation.

Overview of stem cell terminologyThe review includes the following topics:

Stem cells – the basics

Stem cells are unspecialized cells that are characterized by their ability to self-renew and differentiate. They can divide into cells that bear characteristics identical to themselves (self-renewal), or they can change into specialized cells with a more limited developmental potential, (differentiation). Stem cells exist both in embryos and adults. Stem cells that are derived from distinct developmental stages may display different developmental potential:

  • Totipotent stem cells have the potential to generate an entire functional organism, including not only the embryo but also the extra-embryonic tissues. Examples of totipotent cells are  the fertilized eggs and early embryonic cells of mammals.
  • Pluripotent stem cells (PSCs) can give rise to all the cell types of the entire embryo but not the extraembryonic tissues, such as placenta. Examples of pluripotent stem cells include:
    embryonic stem cells (ESCs): derived from the inner cell mass of preimplantation embryos 1, and epiblast stem cells (EpiSCs) derived from the epiblast layer of the implanted embryos 2
  • Induced pluripotent stem cells (iPSCs): generated from somatic cells by reprogramming 3 
  • Multipotent stem cells have the capacity to develop into different cell types within the same cell lineage and are, therefore, also referred to as lineage-specific stem cells or progenitors. An example would be hematopoietic stem cells in bone marrow, which can give rise to all types of blood cell and replenish peripheral blood.

The advantages of using small molecules in stem cell biology

Over recent years, small molecules have emerged as essential tools for understanding and regulating stem cells, and manipulating stem cell fate. They can have wide-ranging effects -  from reprogramming, expansion or directed differentiation of stem cells, to therapeutic effects in in vivo disease models, and survival, ablation, or migration of cancer cells.
Small molecules have advantages over genetic and other methods:

  • they are able to reversibly alter specific functions of a single protein (or multiple proteins) with good temporal control. This is a useful feature, as differentiation into a given lineage is dependent upon a specific sequence of cellular events.
  • they can be used in primary cell assays and easily adapted into in vivo models
  • small molecules can be cell permeable and affect signaling pathways and processes within the cell
  • they are more stable and cost-effective than growth factors
  • small molecules can be of high purity and provide robust reproducible results, with little batch to batch variation in activity
  • the actions of small molecules are concentration dependent – allowing flexibility in the effects achieved
  • Small molecules can be used as chemical probes to further our understanding of the mechanisms that control developmental potential and cell fate.

How can small molecules manipulate stem cell fate?

How can small molecules manipulate stem cell fate?

The adjacent figure shows the processes that can be affected by small molecules and that can modify or manipulate stem cell fate. Small molecules can modify stem cell self-renewal, and induction of lineage-specific differentiation. They can also have an impact on iPSC reprogramming  - they can replace certain transcription factors, enhance the efficiency of reprogramming or accelerate the reprogramming process. The conversion of primed pluripotent stem cells into naive stem cells can also be facilitated by small molecules.

Transdifferentiation  can occur when one somatic cell type converts into another, bypassing pluripotency. It can be mediated either by lineage-specific factors (transdifferentiation I in the figure below) or the restricted reprogramming and subsequent lineage-specific differentiation (transdifferentiation II in the figure below). This latter process can also potentially be affected by small molecules known to be involved in reprogramming and differentiation (see 4 for review)

Use of small molecules for reprogramming

iPS cells have traditionally been generated through exogenous expression of pluripotency genes via viral or episomal vectors but these methods are inefficient, taking weeks to produce small numbers of cells. Additionally, for iPS cell-based therapies to be considered for use in medicine, the use of retroviruses and transcription factors associated with tumorigenesis must also be eliminated. Small molecules can greatly enhance the efficiency of generating iPS cell lines and also reduce or eliminate the need for genetic factors:

For example, PD0325901, Thiazovivin, and SB431542 used in combination, can significantly increase the efficiency of reprogramming human fibroblasts to iPS cells 5.

HDAC inhibitors Valproic acid and Trichostatin A both enhance the yield of iPS cells using the four-factor dedifferentiation method. Using the DNA methyl transferase inhibitor, 5-azacy tidine, can result in a yield 100-fold higher than the four-factor method alone 6.

small molecules used to reprogram mouse fibroblasts without genetic manipulationSubstantial progress has been made in discovering small molecule compounds that maintain pluripotency by functionally replacing one of the distinct reprogramming transcription factors. These molecules can also enhance the efficiency of reprogramming and accelerate the reprogramming process. Valproic acid has also been shown to enhance three-factor reprogramming (minus c-Myc) and two-factor reprogramming (without c-Myc and Klf4) 7.

Small molecules have been shown to be useful in generating high quality iPSCs without genetic defects, preserving the genomic integrity by facilitating the reprogramming process 8.

Reducing the reliance on genetic methods, it is possible to reprogram mouse fibroblasts without the use of any genetic manipulation by using a combination of small molecules including CHIR99021, Forskolin, Tranylcypromine, Valproic Acid, and 3-Deazaneplanocin A9.

Table 1: Small molecules that promote somatic cell reprogramming

M=mouse; H=human

Cat No Name Compound overview Effects on reprogramming Concentration Host animal References
TGFβ receptor inhibitors
HB3301 RepSox (E-616452) TGF-β inhibitor Able to replace Sox2 1 μM M,H 10, 11
HB3218 A 83-01 TGF-β inhibitor Enhances reprogramming 0.5 μM M,H 12, 13
HB3555 SB 431542 TGF-β inhibitor Enhances reprogramming 2 μM M,H 14
GSK-3β inhibitors
HB1261 CHIR 99021 GSK-3β inhibitor and Wnt signaling  activator Able to replace Sox2 3-10 μM M,H 14, 15
HB1266 Kenpaullone GSK-3/CDKs inhibitor Able to replace Klf4 5 μM M 16
MEK inhibitors
HB2240 PD 0325901 MEK inhibitor Enhances reprogramming 0.5-1 μM M,H 17
cAMP agonists
HB1348 Forskolin Adenylyl cyclase agonist. Increases cAMP levels. Able to replace Oct4 (with 2-Me-5HT & D4476)   M 9
HB3460 Prostaglandin E2 Prostaglandin E2 receptor 4 agonist. Increases cAMP levels. Enhances reprogramming 5 μM M 9
Sonic hedgehog signaling activators      
HB3179 JK 184 (Shh antagonist) Sonic hedgehog signaling inhibitor Able to replace Sox2, Klf4, c-Myc 500 ng/ml M 18
Histone deacetylase (HDAC) inhibitors      
HB1399 Sodium butyrate (NaB) HDAC inhibitor Enhances reprogramming 0.5-1 mM M,H 19
HB0867 Valproic acid sodium salt (VPA) HDAC inhibitor Enhances reprogramming. Also able to replace c-Myc/Klf4 in human fibroblasts 0.5-2 mM M,H 20, 7, 15
HB1396 SAHA HDAC inhibitor Enhances reprogramming 5 μM M 20
HB1402 Trichostatin A (TSA) HDAC inhibitor Enhances reprogramming 20 nM M 20
HB1412 Tranylcypromine hydrochloride (Parnate) H3K4 demethylation inhibitor Enhances reprogramming 5-10 μM M 14
HB1356 5-aza-2'-deoxycytidine (Decitabine) DNMT inhibitor Enhances reprogramming 0.5 mM M 21, 22
HB1377 RG 108 DNMT inhibitor Able to replace Sox2 (with BIX 01294) or Oct4 0.04-500 μM M 23, 24, 14
Histone methyltransferase (HMT) inhibitor      
HB1413 BIX 01294 G9a HMTase inhibitor Enhances reprogramming. Also able to replace Oct4 0.5-2 μM M 17
Src family tyrosine kinase inhibitors      
HB1334 PP 1 Src family tyrosine kinase inhibitor Able to replace Sox2 10 μM M 25
HB0758 AMI-5 (Eosin Y) Protein arginine methyltransferase inhibitor Able to replace Sox2, Klf4 (with A-83-01) 5 μM M 13
HB2167 D 4476 CK1 inhibitor Able to replace Oct4 (with Forskolin & 2-Me-5HT) 5 μM M 9
HB2779 Rapamycin mTOR inhibitor Enhances reprogramming 0.3 nM M 26
HB0542 Quercetin Hypoxia inducible factor (HIF) pathway activator Enhances reprogramming 1 μM H 12
HB1209 (±)-Bay K 8644  L-type  Ca2+ channel agonist Able to replace Sox2 2 μM M 23


Effects of small molecules on self-renewal

Maintaining stem cells in defined culture systems is important as it helps to reduce experimental variability. To promote self renewal, it is common practice to add basic FGF into the medium, when culturing human ES cells and culture the cells on a ‘feeder layer’ of mouse embryonic fibroblasts (MEFs) or in conditioned media.  However, the use of serum products and feeder layers has some disadvantages:

  • Feeder layers may restrict the use of human ES cells in therapeutic settings due to concern of xenogenic contamination; increasing the variability of results, and limiting use on a large scale.
  • Batch to batch variability can be a problem when using serum products
  • The use of serum products and feeder layers may also bias stem cell fate toward specific lineage types, through the activation of certain signaling pathways 27 
  • Small molecules can reduce or eliminate the need for serum products and feeder layers. For example:

Pluripotin is a small molecule that promotes the long-term maintenance of mouse ES cells without the need for feeder layers, LIF, BMPs or Wnt proteins 28. ID 8 is another small molecule that promotes mouse ES cell proliferation in serum-free conditions29.

Small molecules such as CHIR99021 (a GSK-3β inhibitor that supresses the Wnt pathway), PD0325901 (which inhibits the MEK pathway), and SB 203580 can stimulate self-renewal of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells30.

Table 2: Small molecules that affect pluripotent stem cell self-renewal and maintenance   

Cat No Name Overview Target Cell
HB1261 CHIR 99021 Inhibits GSK-3. Promotes self-renewal. mESC
HB1259 BIO Inhibits GSK-3. Activates Wnt signaling and maintains ESC self-renewal. hESC and mESC
HB3240 Thiazovivin Inhibits ROCK. Enhances ESC survival. hESC
HB3133 SU 5402 Inhibits FGFRR. Maintains self-renewal. mESC
HB2223 Pluripotin (SC-1) Inhibits RasGAP and ERK1. Promotes self-renewal. mESC
HB1302 SB 203580 Inhibits P38-MAPK. Promotes mESC survival. mESC
HB3282 IQ 1 Binds to PP2A. Decreases p300 phosphorylation and maintains mES cells in an undifferentiated state. mESC


Y-27632 dihydrochlorideSmall molecules can increase the survival of single cells

The low viability of single cells in cultured human pluripotent stem cells makes genome-editing and cloning techniques difficult. ROCK inhibitors Y-27632 32 and Thiazovivin have been shown to increase survival of single human embryonic stem cells through inhibition of RHO/ROCK signaling31.

Small molecules can control differentiation and proliferation

Small molecules provide an effective way of controlling cellular differentiation, reducing the need for more expensive growth factors. By using small molecules to selectively activate and inhibit specific developmental signalling pathways, it is possible to induce differentiation of pluripotent stem cells to specialized cell types.  These developmental pathways include:

The retinoic acid pathway

The synthetic retinoid EC23 is an example of a small molecule, acting through this pathway, that is a potent inducer of stem cell differentiation33.

SB 431542Transforming Growth Factor-β Superfamily

SB431542 is an inhibitor that targets the TGF-β superfamily and is an important tool in stem cell biology – it acts by inhibiting activin receptor-like kinases 4, 5 and 7 (ALK4, TGF-βR1 and ALK7 respectively), and has effects on proliferation, differentiation and promotion of sheet formation of endothelial cells derived from ES cells34. It also promotes differentiation of glioblastoma CS cells11. More recently it has been shown that inhibition of Activin/Nodal/TGF-β and BMP signaling pathways by SB431542 together with Dorsomorphin induces neuronal differentiation of human adipose derived stem cells35.

Canonical Wnt Pathway

This signaling pathway has a significant and well documented role in proliferation, self-renewal and differentiation of stem cells36, 37.  Abberation of this pathway can result in tumourigenesis, due to increased activation leading to increased cellular proliferation.
In terms of small molecules, GSK-3β inhibitor CHIR99021 is commonly used to efficiently direct human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, growth factor- and serum-free system by modulating canonical Wnt signalling38.  IWP-2, another WNT pathway inhibitor induces cardiomyocyte differentiation in pluripotent stem cell-generated mesoderm38.

Kunisada and colleagues developed a protocol for generating insulin-producing cells from hiPS cells and showed that treatment with Activin A and CHIR99021 enhanced efficient endodermal differentiation. They also demonstrated that small molecules Forskolin, Dexamethasone, and SB431542 (a TGF-β inhibitor), were able to induce the differentiation of insulin-producing cells from pancreatic progenitor cells39.

Notch signaling pathway

Notch pathway inhibitor DAPT has recently been shown to promote differentiation of neural stem/progenitor cells into neurons40 and to promote cardiac differentiation of murine pluripotent stem cells41.

Other signaling pathways: Hedgehog and FGFs

The Hedgehog signaling pathway: Hedgehog (Hh) signaling is essential for self-renewal and cell fate determination. Dysfunctional Hh signaling is associated with the development and progression of various types of cancer and is implicated in multiple aspects of tumourigenesis, including the maintenance of cancer stem cells42.  Examples of small molecules that act on Hh signaling pathways include Cyclopamine and GANT61.

Fibroblast growth factors (FGFs) play a key role in the proliferation and differentiation of a variety of cells, and can activate the MAPK/ERK pathway through MEK (MAPK/ERK kinase) activation. PD0325901 a small molecule MEK (MAP3 kinase) inhibitor, promotes the efficiency of mouse and human iPSC (miPSC, hiPSC) reprogramming and late somatic cell reprogramming (after Oct4 activation). PD0325901 also inhibits the growth of non-iPSC colonies and supports the growth of reprogrammed iPSCs43. Small molecule inhibitors or the FGF receptor itself include PD 173074

Table 3: Small molecules that regulate differentiation

Cat No Name Overview Target Cell
HB2266 LY 294002 hydrochloride Inhibits PI3K. Promotes differentiation to mesoderm. hESC and mESC
HB2800 Dorsomorphin dihydrochloride (Compound C) Inhibits BMP. Promotes neural differentiation. hESC and hiPSC
HB1237 Verapamil hydrochloride Blocks L-type Ca2+ channels. Promotes cardiomyocyte differentiation. mESC
HB3011 Stauprimide Inhibits NME2 nuclear localization and downregulates c-Myc. Enhances endoderm, ectoderm and mesoderm differentiation. mESC and hESC
HB0002 (-)-Indolactam V Activates PKC signaling. Promotes pancreatic differentiation. hESC and mESC


Targeting multiple pathways – cocktails of small molecules

In many instances, combining small molecules targeting multiple pathways essential for development has been shown to be an effective approach for inducing differentiation. For example, a combination of SB431542, LDN-193189, CHIR99021, and SU5402 (inhibiting FGFR and VEGFR), and DAPT (inhibiting γ-secretase) induced differentiation of hPSCs into nociceptor neurons in a much accelerated manner with >75% efficiency within 10 days44.


To summarise, the development and use of small molecules is an exciting branch of stem cell biology – offering the researcher opportunities to improve reprogramming efficiency, self-renewal, control differentiation and proliferation as well as providing crucial insights into the signaling and epigenetic mechanisms that regulate stem cell biology.


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