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Stem cells are defined by their ability to differentiate into specialized cells and to self-renew. Pluripotent stem cells (PSCs), of which embryonic stem cells (ESCs) are an example, can differentiate into almost all cell types within the body. Somatic, or adult, cells such as fibroblasts can also be reprogrammed to generate induced pluripotent stem cells (iPSCS).
Typically, reprogramming of somatic cells and differentiation of PSCs into terminal lineages was achieved through exogenous gene expression, via retroviral transfer. This method is inefficient, with production of a relatively small number of cells taking weeks. Additionally, viral vectors must be carefully selected and tested as they have the potential to introduce genetic material or mutations into the cell genome and to be tumorigenic.
Using small molecules for reprogramming and differentiation, as well as maintenance and proliferation of cells in culture has advantages over these methods.
Compared to exogenous gene expression methods, small molecules have effects within hours and greatly reduce the time associated with reprogramming and differentiation. For example, a small molecule cocktail, including SB 431542 (Cat.No. 1614), LDN 193189 (Cat.No. 6053), XAV 939 (Cat.No. 3748), PD 0325901 (Cat.No. 4192), SU 5402 (Cat.No. 3300) and DAPT (Cat.No. 2634) has been shown to differentiate iPSCs into functional cortical neurons within 16 days (Qi et al, 2017).
Small molecules are chemically produced, in contrast to proteins such as growth factors, which are manufactured via biological means. Small molecules therefore have a high level of purity and low batch to batch variation, ensuring consistent activity and reproducible results when used in stem cell culture.
Small molecules are cell permeable, so can be used to target intracellular signaling pathways in both in vitro cell culture and in vivo. They also have concentration-dependent actions and can therefore be used in multiple protocols with different outcomes. For example, CHIR 99021 (Cat.No. 4432) can be used at 20 µM to transdifferentiate fibroblasts into neurons (Li et al, 2015) and at 3 µM to maintain a population of murine ESCs (Kolodziejczyk et al, 2015).
Given their rapid and reversible effects, small molecules can target a protein (or multiple proteins) with high temporal control. This is particularly important in protocols where the effects of a small molecule are required for a specific time period.
Exogenous gene expression using viral vectors has the potential to introduce unwanted genetic material, but animal-free small molecules are devoid of this possibility. Considering the therapeutic potential of iPSC-derived cell therapies, the safety of small molecule use is very important.
Kolodziejczyk et al. (2015) Single cell RNA-sequencing of pluripotent states unlocks modular transcription variation. Cell Stem Cell. 17, 471. PMID: 26431182
Li et al. (2015) Small-molecule driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell. 17, 195. PMID: 26253201
Qi et al. (2017) Combined small-molecule inhibition accelerates the derivation of functional, early-born, cortical neurons from human pluripotent stem cells. Nat. Biotechnol. 35, 154. PMID: 28112759