Controlling the Differentiation and Growth of Stem Cells with Small Molecules

Last updated: 04-17-2020

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Controlling the Differentiation and Growth of Stem Cells with Small Molecules

During the in vitro culture of cells, small molecules are often used to manipulate signaling pathways. These signaling pathways are important targets for small molecules in the culture of stem cells that control cell proliferation and differentiation. It can be useful to target pathways such as the canonical Wnt, transforming growth factor-β (TGF-β) and retinoic acid signaling pathways  to increase and maintain the proliferation of stem cells. Alternatively, this can be done to guide stem cell fate toward specific lineages in controlled differentiation.

This article offers a brief insight into the small molecules that interact with the primary signaling pathways which govern stem cell proliferation and differentiation to mediate stem cell behavior, as well as how small molecules play certain roles in the dedifferentiation of somatic cells to create populations of pluripotent stem cells.

The defining factor of stem cells is their ability to self-renew and their potential to differentiate into defined cellular subtypes. Four main types of stem cell exist; embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, adult stem (AS) cells and cancer stem (CS) cells. Historically, ES cells derive from the inner cell mass of the developing blastocyst.

Upon exposure to developmental cues, ES cells can differentiate into any cell type, representing all three of the developing germ layers. Useful therapeutic tools and insight into key developmental processes may be provided by the study of ES cells. Through either forced expression of transcription factors, or exposure to a multitude of molecules that revert them back to a stem cell-like phenotype, iPS cells are derived from the reprogramming of somatic cells.

iPS cells are thought to have a similar potency to ES cells, as they are both pluripotent and able to differentiate into cell types representing all three germ layers. This may provide therapeutic potential as treatment for degenerative diseases for autologous transplantation of iPS cell-derived cell types.

AS cells are conventionally responsible for the maintenance and repopulation of cell types found within specific niches in tissues, and have a much more limited differentiation potential. For instance, the hematopoietic stem cell found inside the bone marrow, that can give rise to only cell types found within the blood is an AS cell.

The CS cell, the final category of stem cell, is responsible for the proliferation of cells in specific kinds of tumor. These  cells may constitute a potential therapeutic target for anti-cancer drug development as they are thought to be implicated in cancer progression, initiation and metastasis. An integral part of stem cell research are the synthetic and naturally occurring molecules that interact with certain signaling pathways.

One can use compounds that are designed to interact with specific stages in developmental pathways to evoke a particular cellular response, which can then be modulated through compound concentration changes. The controlled differentiation of stem cells into specific cellular lineage can be achieved by the selectivity of molecules to act only upon the desired pathway. This is vital for in vitro modeling, which is used for basic research, drug screening and the study of pathological mechanisms. Two approaches which are frequently used to discover small molecules that interact with key signaling pathways are the custom design of molecular structures and screening of compound libraries.

Several approaches can be used to identify small molecules that manipulate cell fate (reviewed by Lyssiotis et al). One of the most prevalent methods is high-throughput cell-based phenotypic screening, which involves the screening of large chemical libraries using immortalized cell lines.

A relatively straightforward example of high-throughput screening are reporter-based cellular assays: these involve the expression of a fluorescent reporter gene that is stimulated by the promoter of the gene of interest. Work by Kumagai et al constitutes an example of the use of reporter-based assays in stem cell research. They are screened for small molecules that promote human ES cell self-renewal to retain populations of undifferentiated cells.

The key processes involved included observing green fluorescent protein expression driven by the Oct4 promoter, a transcription factor and marker of pluripotency,  to determine how or if the small molecules tested affected cell fate. Though this method is largely well-suited to screening large libraries of molecules, the potential exists for the production of false positives and results still require rigorous testing.

The multi-parametric, high-content image-based assay is another high-throughput screening method of small molecule identification, which involves the analysis of a desired phenotype at a single-cell level. This process is effective, but costly and time consuming.

The size of libraries screened using high-throughput approaches can range from large collections of >2 million compounds (conventionally held by pharmaceutical companies) to smaller libraries of


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