High Throughput Mechanobiological Screens Enable Mechanical Priming of Pluripotency in Mouse Fibroblasts

Transgenic methods for direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) are effective in cell culture systems but ultimately limit the utility of iPSCs due to concerns of mutagenesis and tumor formation. Recent studies have suggested that some transgenes can be eliminated by using small molecules as an alternative to transgenic methods of iPSC generation. We developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. Using this system, we screened for optimized conditions to stimulate the activation of Oct-4 and other transcription factors to prime the development of pluripotency in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the priming of pluripotency of mouse fibroblasts in combination with mechanical loading. Taken together, our findings demonstrate the ability of mechanical forces to induce reprograming factors and support that biophysical conditioning can act cooperatively with small molecules to priming the induction pluripotency in somatic cells.

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A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Scientific Reports ◽

10.1038/s41598-020-72158-5 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jason Lee ◽

Miguel Armenta Ochoa ◽

Pablo Maceda ◽

Eun Yoon ◽

Lara Samarneh ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Cultured Cells ◽

Mechanical Load ◽

Mechanical Stretch ◽

Mechanical Forces ◽

Mouse Fibroblasts ◽

Factor Expression ◽

Physiological Homeostasis ◽

Transcription Factor Expression

Abstract Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

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Direct Reprogramming of Mouse Fibroblasts to Neural Stem Cells by Small Molecules

Stem Cells International ◽

10.1155/2016/4304916 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 23

Author(s):

Yan-Chuang Han ◽

Yoon Lim ◽

Michael D. Duffieldl ◽

Hua Li ◽

Jia Liu ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Neural Stem Cells ◽

Small Molecules ◽

Clinical Medicine ◽

Viral Vector ◽

Expression Patterns ◽

Tumor Formation ◽

Patient Specific ◽

Mouse Fibroblasts

Although it is possible to generate neural stem cells (NSC) from somatic cells by reprogramming technologies with transcription factors, clinical utilization of patient-specific NSC for the treatment of human diseases remains elusive. The risk hurdles are associated with viral transduction vectors induced mutagenesis, tumor formation from undifferentiated stem cells, and transcription factors-induced genomic instability. Here we describe a viral vector-free and more efficient method to induce mouse fibroblasts into NSC using small molecules. The small molecule-induced neural stem (SMINS) cells closely resemble NSC in morphology, gene expression patterns, self-renewal, excitability, and multipotency. Furthermore, the SMINS cells are able to differentiate into astrocytes, functional neurons, and oligodendrocytesin vitroandin vivo. Thus, we have established a novel way to efficiently induce neural stem cells (iNSC) from fibroblasts using only small molecules without altering the genome. Such chemical induction removes the risks associated with current techniques such as the use of viral vectors or the induction of oncogenic factors. This technique may, therefore, enable NSC to be utilized in various applications within clinical medicine.

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Dot1L methyltransferase activity is a barrier to acquisition of pluripotency but not transdifferentiation

10.1101/835413 ◽

2019 ◽

Cited By ~ 1

Author(s):

Coral K. Wille ◽

Rupa Sridharan

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Somatic Cells ◽

Cell Types ◽

Transcriptional Elongation ◽

Mrna Levels ◽

Mesenchymal To Epithelial Transition ◽

Ipsc Generation ◽

Induced Pluripotent

ABSTRACTThe ability of pluripotent stem cells to be poised to differentiate into any somatic cell type is partly derived from a unique chromatin structure that is depleted for transcriptional elongation associated epigenetic modifications, primarily H3K79 methylation. Inhibiting the H3K79 methyltransferase, Dot1L, increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) most potently at the mid-point of the process. Surprisingly, despite the enrichment of H3K79me2 on thousands of actively transcribed genes, Dot1L inhibition (Dot1Li) results in few changes in steady state mRNA levels during reprogramming. Dot1Li spuriously upregulates genes not involved in pluripotency and does not shutdown the somatic program. Depletion of the few genes that are downregulated, such as Nfix, enhances reprogramming efficiency in cooperation with Dot1Li. Contrary to the prevalent view, Dot1Li promotes iPSC generation beyond early phases of reprogramming such as the mesenchymal to epithelial transition and from already epithelial cell types including keratinocytes. Significantly, Dot1L inhibition does not enhance lineage conversion to neurons or muscle cells. Taken together, our results indicate that H3K79me is not a universal barrier of cell fate transitions but specifically protects somatic cells from reverting to the pluripotent state.

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Using Small Molecules to Improve Generation of Induced Pluripotent Stem Cells from Somatic Cells

Cellular Programming and Reprogramming - Methods in Molecular Biology ◽

10.1007/978-1-60761-691-7_13 ◽

2010 ◽

pp. 207-218 ◽

Cited By ~ 36

Author(s):

Caroline Desponts ◽

Sheng Ding

Keyword(s):

Stem Cells ◽

Small Molecules ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Somatic Cells ◽

Induced Pluripotent

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Molecular Mechanisms of Induced Pluripotency

Acta Naturae ◽

10.32607/20758251-2012-4-1-12-22 ◽

2012 ◽

Vol 4 (1) ◽

pp. 12-22 ◽

Cited By ~ 9

Author(s):

I. A. Muchkaeva ◽

E. B. Dashinimaev ◽

V. V. Terskikh ◽

Yu. V. Sukhanov ◽

A. V. Vasiliev

Keyword(s):

Transcription Factors ◽

Small Molecules ◽

Molecular Mechanisms ◽

Somatic Cells ◽

Ips Cells ◽

Direct Reprogramming ◽

Induced Pluripotency ◽

Somatic Cell Reprogramming ◽

Induced Pluripotent ◽

Genome Modifications

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered.

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Reprogramming with Small Molecules instead of Exogenous Transcription Factors

Stem Cells International ◽

10.1155/2015/794632 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 34

Author(s):

Tongxiang Lin ◽

Shouhai Wu

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Small Molecules ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Mouse Embryonic Fibroblast ◽

Patient Specific ◽

Full Potential ◽

Ipsc Generation ◽

Induced Pluripotent

Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

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Faculty Opinions recommendation of Rapid and efficient reprogramming of somatic cells to induced pluripotent stem cells by retinoic acid receptor gamma and liver receptor homolog 1.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13371021.14747117 ◽

2011 ◽

Author(s):

John Gurdon ◽

Vincent Pasque

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Retinoic Acid Receptor ◽

Somatic Cells ◽

Acid Receptor ◽

Induced Pluripotent

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STEM-20. A CANCER STEM CELL-SELECTIVE APOPTOSIS-INDUCING SMALL MOLECULE FOR THE TREATMENT OF GBM

Neuro-Oncology ◽

10.1093/neuonc/noaa215.837 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii200-ii200

Author(s):

Stephen Skirboll ◽

Natasha Lucki ◽

Genaro Villa ◽

Naja Vergani ◽

Michael Bollong ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Small Molecules ◽

Small Molecule ◽

Large Scale ◽

Critical Role ◽

Tumor Formation ◽

Cell Type ◽

Caspase 1 ◽

Activation Assay

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

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Vasopressin-vermittelte Wasserrückresorption im Sammelrohr der Niere

BIOspektrum ◽

10.1007/s12268-021-1544-1 ◽

2021 ◽

Vol 27 (2) ◽

pp. 165-167

Author(s):

Sandrine Baltzer ◽

Enno Klussmann

Keyword(s):

Water Balance ◽

Small Molecules ◽

High Throughput ◽

Molecular Mechanisms ◽

Collecting Duct ◽

Water Channel ◽

Water Reabsorption ◽

Balance Disorders ◽

Water Homeostasis ◽

Balance Disorder

AbstractVasopressin-mediated water reabsorption from primary urine in the renal collecting duct is essential for regulating body water homeostasis and depends on the water channel aquaporin-2 (AQP2).Dysregulation of the process can cause water balance disorders. Here, we present cell-based high-throughput screenings to identify proteins and small molecules as tools to elucidate molecular mechanisms underlying the AQP2 control and as potential starting points for the development of water balance disorder drugs.

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