Defined by their ability to differentiate into specialized cells, stem cells are also characterized by their capacity to self-renew. For instance, Pluripotent stem cells (PSCs), in particular, embryonic stem cells (ESCs), can differentiate into almost all cell types within the body. In addition, somatic (also known as adult) cells, such as fibroblasts, have the capability to be reprogrammed and thus generate induced pluripotent stem cells (iPSCS).
In the past, the reprogramming of somatic cells and subsequent differentiation of PSCs into terminal lineages was accomplished through exogenous gene expression, via a process known as retroviral transfer.
An inefficient process, this method resulted in the production of a relatively small number of cells which took weeks. What’s more, the viral vectors require careful selection and testing, due to their potential for introducing genetic material or mutations into the cell genome and for being tumorigenic.
The use of small molecules in the process of reprogramming, differentiation, as well as cell maintenance and proliferation in culture, has several advantages over the above methods:
Cost Effective, Quick and Convenient Small Molecules
In comparison to methods of exogenous gene expression, small molecules produce effects within hours and significantly reduce the time associated with reprogramming and differentiation. For instance, a small molecule cocktail, containing SB 431542, LDN 193189, XAV 939, PD 0325901, SU 5402 and DAPT demonstrated the ability to differentiate iPSCs into functional cortical neurons within 16 days (Qi et al, 2017).
Small Molecules are Synthetically Produced
In contrast to proteins, like growth factors manufactured through biological means, small molecules are chemically produced. Thus, small molecules come with a high level of purity and low variation from batch-to-batch, ensuring consistent activity and reproducible results when used in stem cell culture.
Small Molecules are Cell Permeable and Tuneable
Small molecules are cell permeable, so can be used to target intracellular signaling pathways in both in vitro cell culture and in vivo. Small molecules also have concentration-dependent actions. As a result, they can be used in multiple protocols with different outcomes. An example of this is CHIR 99021, which 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).
Small Molecules have Good Temporal Control
Small molecules can target a protein (or multiple proteins) with high temporal control, owing to their rapid and reversible effects. Such a facet is particularly vital in protocols in which the effects of a small molecule are needed for a specific time period.
Small Molecules are Safe
The use of viral vectors for exogenous gene expression has the potential to introduce unwanted genetic material. However, animal-free small molecules are devoid of this capability. 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(4), 471.
- Li et al. (2015) Small-molecule driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell. 17 (2), 195
- 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 (2), 154.
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