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Stem cell therapies are entering the clinic, and with the clinical phases of development being highly regulated, there is a requirement for ancillary reagents (raw materials) to meet Good Manufacturing Practice (GMP) standards, to assure therapy safety and suitability.

Product Name Catalog # Action

CHIR 99021 TB4423-GMP Potent and Selective GSK-3 Inhibitor;

WNT Pathway Activator

LDN 193189 NEW TB6053-GMP Potent and Selective ALK2 and ALK3 Inhibitor. Commonly used with SB 431542 (dual SMAD inhibition)

SB 431542 TB1614-GMP TGF-b1, ALK4 and ALK7 Inhibitor. Commonly used with LDN 189193 (dual SMAD inhibition)

XAV 939 TB3748-GMP Potent Tankyrase Inhibitor;

Inhibits WNT Signaling

Y-27632 dihydrochloride TB1254-GMP Selective ROCK Inhibitor. Commonly used to improve survival during passaging and freezing of cell cultures

GMP Small Molecules

When Is The Right Time To Go GMP?

Are you looking to translate your research into clinical applications? As you advance your research towards clinical manufacturing, you should have confidence that incorporating animal-free and GMP raw materials is efficient and does not functionally impact your process. By switching early to GMP you can reduce the risk, time and cost associated with re-validating assays and protocols, which may be required when switching at a later stage.

Find out more: tocris.com/product-type/gmp-small-molecules

What Is GMP?

• GMP (or cGMP; Current Good Manufacturing Practice) is a set of guidelines that ensure safe, reliable, consistent and quality-assured products. GMP small molecules are manufactured with increased process control, are subject to enhanced quality control testing, and are supplied with more complete documentation to ensure appropriate quality control

• Evidence must be provided throughout the manufacturing process to prove GMP requirements have been met

• The product is GMP only if the processes and controls around the product’s manufacture are proven to meet GMP standards

STRY0262205_SMBU_FL_GMP-Grade _GP

Why Choose Bio-Techne As Your Raw Materials Supplier?

Our experience as a raw materials supplier ensures:

• ISO 9001:2015 certified Quality Management System

• Batch-to-batch consistency

• Reliable supply

Stem Cell Resources

For further information on Good Manufacturing Practices at Bio-Techne visit: bio-techne.com/gmp-products/gmp-capabilities

Stem Cell Research Product Guide

Our research product guide highlights the use of small molecules in stem cell research and cell therapy.

This poster summarizes some key protocols demonstrating the use of small molecules across the stem cell workflow.

This review summarizes the use of small molecules in controlling stem cell growth and differentiation.

Scientific Poster: Stem Cell Workflow

Scientific Review: Stem Cell Growth and Differentiation

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Somatic Cell Isolation

Clinical Applications

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 cell ROCK 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. 17 1079 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

Products available from Tocris Reprogramming CHIR 99021 PD 0325901 SB 431542 A 77-01 3-Deazaneplanocin A (S)-(+)-Dimethindene DBZ Thiazovivin (±)-Bay K 8644 Trichostatin A RepSox Valproic acid, sodium salt Kenpaullone Alsterpaullone SMER 28 L-Ascorbic acid BIX 01294 Crotonic Acid Tranylcypromine

Differentiation LDN 193189 XAV 939 DAPT SAG dihydrochloride Dibutyryl-cAMP, sodium salt Forskolin SU 5402 IWP 2 IWP 4 IDE 1 IBMX Fluoxetine Metformin Dorsomorphin Wnt-C59 1-EBIO ISX 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

Reprogramming is the regression of a specialized cell to a simpler state resulting in cells with stem-like properties known as induced pluripotent system cells (iPSCs). Takahashi et al. (2007) first reported reprogramming of specialized human adult cells by introducing the transcription 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.

Small molecules can be used to enhance reprogramming efficiency: Valproic acid (VPA) can increase reprogramming efficiency by Yamanaka factors by >100 fold. Using a cocktail of small molecules and growth factors to reprogram cells dispenses with the need for retroviral 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 (see Self-Renewal panel), could be used to reprogram mouse somatic cells at a frequency of 0.2% (compared with 0.01 – 0.02% by the Takahashi method).

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 to generate 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.

Reprogramming

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

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 formation of 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.

Verification

Stem cells can be differentiated into numerous cell types with a variety of potential uses, including drug screening, toxicity testing and disease 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.

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.

Differentiation

Stem cells self-renew and proliferate by the division of a parent cell into two identical daughter cells. Conventional culture methods for PSCs require ‘feeder’ cells, serum products and growth factors, such as LIF and bFGF. More recently, chemically-defined serum-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 of stem cells.

Chen et al. were the first to show that a small molecule, SC 1 (also known as Pluripotin), which inhibits differentiation, can be used to maintain pluripotency in the absence of growth factors. Ying et al. (2008) subsequently identified a combination of 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).

Self-Renewal

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 the ROCK inhibitor Y-27632 prior to freezing, significantly increases the viability of cells after thawing. Y-27632 also improves the survival of cells that have been differentiated from PSCs, facilitating their use in disease modeling and therapy.

Storage

Cells 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 into cardiomyocytes 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.

Qi 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.

3-Deazaplanocin AVC6TF

Day 0 Day 16–20 Day 28–36 Mouse embryonic fibroblasts ciPSC colonies

Chemical Reprogramming Medium

Mouse embryonic fibroblasts

XEN-like colonies CiPSC colonies

VC6TF, Ch 55 EPZ 004777

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

Optimized stage I medium

Optimized N2B27-SII medium supplemented with LIF, Ascorbic acid and bFGF

Stage III medium supplemented with CHIR 99021, PD 0325901, LIF and Ascorbic acid

Day 0 Day 4–6 Day 20+

Fibroblasts Cardiomyocytes Reprogramming medium Cardiac induction medium Infarcted mouse

9C Cocktail

hPSCs Pancreatic progenitors Functional β-cells

Primitive gut tube formation

3 days

Day 0 Day 27+

3 days 7 days 7–14 days

DE induction Pancreatic specification β-cell maturation

hiPSCs

FGF2, Y-27632 LDN 193189, SB 431542, XAV 939

PD 0325901, SU 5402, DAPT Immature cortical neurons

Day 0 Day 8

hiPSCs Chemically-induced neurons

Application of neurotransmitters, antagonists, toxins, etc

0 mV

2i

Can be maintained indefinitely in vitro

ESCs or iPSCs Freeze Thaw

Y-27632

For copies of this poster, please visit tocris.com © 2019 Tocris Cookson, Ltd. Tocris is a Bio-Techne brand

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 be used 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, their effects are rapid and reversible, so their use can reduce the duration of reprogramming and differentiation protocols.

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Stem Cell Workflow: Using Small Molecules

Contents Introduction ............................................................................................. 1

Identification of Small Molecules ......................................................... 2

Pathways that Modulate Stem Cell Activity ............................................2

Retinoic Acid ...................................................................................... 2

Hedgehog Pathway ........................................................................... 3

Transforming Growth Factor-b Superfamily ..................................... 4

Canonical Wnt Pathway .................................................................... 5

Fibroblast Growth Factor and Notch Signaling Pathways ............... 8

Promotion of ES Cell Self-Renewal ..........................................................9

Somatic Cell Reprogramming ............................................................... 10

Conclusion ................................................................................................ 10

References ............................................................................................... 11

Stem Cell Compounds ............................................................................12

Introduction Small molecules are routinely used to manipulate signaling pathways during the in vitro culture of cells. Signaling pathways that control cell proliferation and differentiation are important targets for small molecules in the culture of stem cells. Targeting pathways such as the canonical Wnt, transforming growth factor-b (TGF-b) and retinoic acid signaling pathways can be useful to enhance and maintain the proliferation of stem cells, or to guide stem cell fate toward specific lineages in controlled differentiation. This review provides a brief overview of the small molecules that interact with the primary signaling pathways that govern stem cell proliferation and differentiation to mediate stem cell behavior, along with the role of small molecules in the dedifferentiation of somatic cells to create populations of pluripotent stem cells.

Stem cells are characterized as having the ability to self-renew along with the potential to differentiate into defined cellular subtypes.1 There are four main types of stem cell; embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, adult stem (AS) cells and cancer stem (CS) cells. ES cells are historically the most potent and are derived from the inner cell mass of the developing blastocyst. They are able to differentiate into any cell type representing all three of the developing germ layers upon exposure to developmental cues. The study of ES cells may provide useful therapeutic tools and insight into key develop- mental processes.2 iPS cells are derived from the reprogramming of somatic cells through forced expression of transcription factors, or exposure to a multitude of molecules that revert them back to a stem cell-like phenotype.3 They are thought to have a similar potency to ES cells as they are pluripotent and able to differentiate into cell types representing all three germ layers. This may provide therapeutic potential for autologous transplantation of iPS cell-derived cell types as treatment for degenerative diseases.

AS cells have a much more restricted differentiation potential and are typically responsible for the maintenance and repopulation of cell types found within specific niches in tissues. An example of an AS cell is the hematopoietic stem cell found within the bone marrow that can give rise to only cell types found within the blood.4 The final category of stem cell, the CS cell, is responsible for cell proliferation within certain types of tumor. CS cells are thought to be implicated in cancer progression, initiation and metastasis, and therefore may provide a potential therapeutic target for anti-cancer drug development.5

Synthetic and naturally occurring molecules that interact with certain signaling pathways are an integral component of stem cell research. Compounds designed to interact with specific stages in developmental pathways can be utilized to invoke specific cellular responses, which can be modulated through changes in compound concentration. The selectivity of molecules

Kirsty E. Clarke1, Victoria B. Christie1, Andy Whiting2 and Stefan A. Przyborski1 1Biological Sciences and 2Department of Chemistry, Durham University, South Road, Durham, DH1 3LE, UK

Correspondence e-mail: stefan.przyborski@durham.ac.uk

Kirsty Clarke and Victoria Christie are research scientists within the laboratory of Professor Stefan Przyborski at Durham University. Research in Professor Przyborski’s group focuses on the development and application of technology to improve the growth, differentiation and function of cultured cells, including the use of small molecules that control stem cell fate.

Professor Whiting is an organic chemist and a long-standing collaborator of Professor Przyborski, working on the design and development of small molecules to control cellular behavior.

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Tocris Scientific Review Series

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Using Small Molecules to Control

Stem Cell Growth and Differentiation

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