Stem Cell Workflow: Using Small Molecules
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Stem cells (SCs) have immense potential as a limitless source of cells and tissues for research and treatment of various diseases, as well as for investigating early embryonic development. Small molecules can beused at all stages of the stem cell work flow, and their use has several advantages over standard techniques: small molecules are chemically-synthesized, versatile, animal-free, and are cell permeable. In addition, theireffects are rapid and reversible, so their use can reduce the duration of reprogramming and differentiation protocols.
Reprogramming

Self-Renewal

Storage

Reprogramming is the regression of a specialized cell to a simpler state resulting in cells with stem-like properties known as induced
pluripotent stem cells (iPSCs). Takahashi et al. (2007) first reported reprogramming of specialized human adult cells by introducing thetranscription factors Oct3/4, Sox2, Klf4 and c-Myc (known as the Yamanaka factors or OSKM) into adult human dermal fibroblasts via
retroviral transduction. The resulting iPSCs had similar properties to ESCs and could differentiate into all three embryonic germ layers.
Similar techniques are still widely used.

Stem cells self-renew and proliferate by the division of a parent cell into two identicaldaughter cells. Conventional culture methods for PSCs require ‘feeder’ cells, serum
products and growth factors, such as LIF and bFGF. More recently, chemically-definedserum-free media have been developed to replace the requirement for feeder cells
and serum products, and small molecules can be used to maintain self-renewal ofstem cells.

Cryopreservation is used to store cells,
including stem cells, for in vitro culture,
however after freeze-thawing the survival
rate of cells can be poor. Li et al. (2008)
found that treatment of hESCs with theROCK inhibitor Y-27632 prior to freezing,
significantly increases the viability of cells
after thawing. Y-27632 also improves
the survival of cells that have beendifferentiated from PSCs, facilitating theiruse in disease modeling and therapy.

Small molecules can be used to enhance reprogramming efficiency: Valproic acid (VPA) can increase reprogramming efficiency byYamanaka factors by >100 fold. Using a cocktail of small molecules and growth factors to reprogram cells dispenses with the need forretroviral transduction and increases the efficiency of reprogramming. Hou et al. (2013) first described the generation of iPSCs using
only small molecules. The researchers discovered that a combination of six compounds, VPA, CHIR 99021, RepSox, Tranylcypromine,
Forskolin (these 5 compounds are together known as VC6TF) and 3-Deazaneplanocin A, followed by culture in 2i medium (seeSelf-Renewal panel), could be used to reprogram mouse somatic cells at a frequency of 0.2% (compared with 0.01 – 0.02% by theTakahashi method).
3-Deazaplanocin AVC6TF

Chen et al. were the first to show that a small molecule, SC 1 (also known asPluripotin), which inhibits differentiation, can be used to maintain pluripotency in theabsence of growth factors. Ying et al. (2008) subsequently identified a combinationof two small molecules (known as 2i), the MEK inhibitor PD 0325901 and GSK-3
inhibitor CHIR 99021, which can sustain ESC self-renewal. ESCs grown in 2i medium
show lower levels of spontaneous differentiation compared with standard SC culture
methods. 2i can also be used to rescue cultures that have started to deteriorate.

The ROCK inhibitor Y-27632 also has an important role in stem cell maintenance as it increases cloning efficiency of ESCs without affecting
pluripotency, enabling the survival of stem cells in culture over the long-term (Watanabe, 2007).
Chemical Reprogramming Medium
Mouse embryonic fibroblasts

Day 0Day 16–20Y-27632

Day 28–36

ciPSC colonies

2i

Somatic Cell
Isolation
FreezeESCs or iPSCs

Thaw

Modification of this protocol by Zhao et al. (2018) led to a highly efficient method for generating ciPSCs from mouse embryonic
fibroblasts (MEFs). The three-stage process uses 12 compounds (including VC6TF) plus the growth factors, LIF and bFGF togenerate ciPSCs in 16 to 20 days compared with 30 days using Yamanaka factors. This type of chemical reprogramming holds
promise for the generation of cells for cell therapy and disease modeling.
VC6TF, Ch 55
EPZ 004777

Can be maintainedindefinitely in vitro

VC6TF, Ch 55, 3-Deazaneplanocin A,Decitabine, SGC 0946

Differentiation
Stem cells can be differentiated into numerous cell types with a variety of potential uses, including drug screening, toxicity testing anddisease modeling. They also hold promise for treating conditions such as neurodegenerative diseases, diabetes, and traumatic injury,
among others. Small molecules are versatile tools to control stem cell fate and direct differentiation toward specific cell types.

Mouse embryonic
fibroblasts

XEN-like colonies
Optimizedstage I medium

Day 0

CiPSC colonies
Optimized
N2B27-SII medium
supplementedwith LIF, Ascorbic
acid and bFGF

Stage III medium
supplementedwith CHIR 99021,
PD 0325901, LIF
and Ascorbic acidDay 4–6

ClinicalApplications

A method to derive functional human pancreatic β-cells from hPSCs designed by Pagliuca et al. (2014) presents the possibility of a new way
to treat diabetes. Their six-stage protocol uses a combination of 11 small molecules and proteins (Activin A, CHIR 99021, Retinoic Acid,SANT-1, LDN 193189, Phorbol 12,13-dibutyrate, T3, Compound E, RepSox, Heparin, Betacellulin and KGF) and produces functional
β-cells in 28 days, which when transplanted into diabetic mice are found to ameliorate hyperglycemia.

Day 20+hPSCsFunctional β-cells
Day 27+

Pancreatic progenitors

Day 0

Primitive gut
DE induction tube formation

Verification
The characterization of differentiated cells is a necessary step before the cells can be used in assays or therapy. Immunohistochemistry orWestern blotting, using antibodies against specific markers expressed by the target cell, are techniques widely used to verify the identityof differentiated cells, as is quantitative RT-PCR, which is used to analyze gene expression. Small molecules play an important part in theverification of cell function, by pharmacological or electrophysiological techniques.

0 mV
Application ofneurotransmitters,
antagonists, toxins, etc

β-cell maturation

7 days

7–14 days

3 days3 daysQi et al. (2017) have developed a protocol using a cocktail of six small molecules to derive cortical neurons from hiPSCs. When transplanted
into postnatal mouse cortex at day 8 of differentiation the resulting neurons are functional and establish long term connections. In addition,
cells remaining in culture exhibit functional electrophysiological properties by day 16.

Following conversion of fibroblasts into cardiomyocytes, Cao et al. established the presence of genes involved in cardiomyocyte function.
Electrophysiological analysis of the cells revealed ventricular-like action potentials. The researchers then looked at the effect of small
molecules on action potential firing and found that Caffeine and the non-selective β-adrenoceptor agonist Isoprenaline increased firing
rate, while the muscarinic agonist Carbachol slowed the firing rate, helping to establish the identity of the differentiated cells as functioning
cardiomyocytes.
Telezhkin et al. (2016) describe the verification of functional neurons differentiated from iPSCs. Electrophysiological analysis revealed
spontaneous action potential firing, which was eliminated by application of Tetrodoxin, indicating the presence of functioning sodium
channels. Application of GABA induced inhibitory postsynaptic currents that could be blocked by Bicuculline, indicative of the formationof a functioning neural network. Calcium imaging using FURA-2AM, revealed Ca2+-influx in response to application of neurotransmitters
GABA, Glutamate or Glycine. Techniques such as these provide evidence for the presence of mature functioning cells.

Pancreatic specification

FGF2, Y-27632

LDN 193189, SB 431542, XAV 939

hiPSCsPD 0325901, SU 5402, DAPTDay 0Immature cortical neurons
Day 8Cells can also be reprogrammed directly from one specialized cell type to another, without first being converted to ciPSCs, a process
known as transdifferentiation or direct-lineage reprogramming. Cao et al. (2016) described a method to convert human fibroblasts intocardiomyocytes using a cocktail of nine small molecules: CHIR 99021, A83-01, SC1, OAC-2, Y-27632, BIX 01294, A 8351, SU 16f
and JNJ 10198409 (9C). The 9C-treated cells were subsequently cultured in cardiac induction medium and transplanted into mice
where they converted into cardiomyocyte-like cells.
9C Cocktail

hiPSCsChemically-induced neurons

Fibroblasts

Reprogramming medium

Cardiomyocytes

Cardiac induction medium

Infarcted mouse

Products available from TocrisReprogramming
CHIR 99021
PD 0325901
SB 431542
A 77-01
3-Deazaneplanocin A(S)-(+)-Dimethindene
DBZ
Thiazovivin(±)-Bay K 8644
Trichostatin A
RepSoxValproic acid, sodium salt
Kenpaullone
Alsterpaullone
SMER 28
L-Ascorbic acid
BIX 01294
Crotonic Acid
Tranylcypromine
Differentiation
LDN 193189
XAV 939DAPT
SAG dihydrochloride
Dibutyryl-cAMP, sodium salt
Forskolin
SU 5402
IWP 2
IWP 4
IDE 1
IBMX
Fluoxetine
Metformin
Dorsomorphin
Wnt-C59
1-EBIOISX 9
Dexamethasone
Zebularine
Proliferation and Cell Viability
Y-27632
A 83-01
Prostaglandin E2
SB 202190
Epiblastin A
MB 05032
U0126
Go 6983
SB 203580
A 769662
LY 294002
CH 223191
Pluripotin
SB 216763
BIO
PD 98059
PD 173074
Troglitazone
Cyclopamine
Mitomycin C
GMP Small Molecules
Y-27632
CHIR 99021
SB 431542
DAPT
Abbreviations
bFGF
Basic fibroblast growth factor
ciPSC Chemically-induced
pluripotent stem cell
ESC
Embryonic stem cell
GSK
Glycogen synthase kinase 3
KGF
Keratinocyte growth factor
LIF
Leukemia inhibitory factor
MEK
Mitogen-activated protein
kinase kinase (MAP2K)PSC
Pluripotent stem cellROCK Rho-kinase
RT-PCR Reverse transcription
polymerase chain reaction
T3
Triiodothyronine
XEN
Extraembryonic endoderm
References
Cao, N et al. (2016) Science 352 6290
Chen, S et al. (2006)
Proc.Natl.Acad.Sci.USA 103 17266
Hou, P et al. (2013) Science 341 651
Ichikawa, H et al. (2011) Cryo Letters
32 516
Li, X et al. (2008) Stem Cells Dev. 171079
Pagliuca, FW et al. (2014) Cell 159 428
Takahashi, K et al. (2007) Cell 131 861
Tamm, C et al. (2013) PLoS One
8 e81156
Telezhkin, V et al. (2016)Am.J.Physiol.Cell Physiol. 310 C520
Watanabe, K et al. (2007)
Nat.Biotechnol. 25 681
Wilson, HK et al. (2016) Tissue Eng.
Part C, Methods 22 1085
Ying, QL et al. (2008) Nature 453 519
Zhao, T et al. (2018) Cell Stem Cell 23 31

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