A secreted proteomic footprint for stem cell pluripotency

With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the spent medium from cultured embryonic (Man-13) and induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of 4 E8- against 4 E6- enriched biomarkers provides 16 ratio abundances which are each robustly diagnostic for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to recovery assays.

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Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

Molecular Biology of the Cell ◽

10.1091/mbc.e19-08-0447 ◽

2020 ◽

Vol 31 (23) ◽

pp. 2583-2596

Author(s):

John M. Boyle ◽

Kelsey M. Hennick ◽

Samuel G. Regalado ◽

Jacob M. Vogan ◽

Xiaozhu Zhang ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Telomere Length ◽

Pluripotent Stem Cells ◽

Human Stem Cells ◽

Set Point ◽

Hypomorphic Allele ◽

Point Control ◽

Set Point Control ◽

Stem Cell Lines

To better understand telomere length set point control in human stem cells, we generated knockout stem cell lines for TPP1 and contrasted their phenotypes with those of homozygous TPP1 L104A mutant stem cells. This comparison reveals that TPP1 L104A is not a hypomorphic allele but formally establishes TPP1 L104 as a dissociation of function mutant.

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Reconstructing human early embryogenesis in vitro with pluripotent stem cells

10.1101/2021.03.12.435175 ◽

2021 ◽

Author(s):

Berna Sozen ◽

Victoria Jorgensen ◽

Meng Zhu ◽

Tongtong Cui ◽

Magdalena Zernicka-Goetz

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Human Development ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Early Embryo ◽

Human Stem Cells ◽

Amniotic Cavity ◽

Early Human

ABSTRACTUnderstanding human development is of fundamental biological and clinical importance. Yet despite its significance, insights into early developmental events in humans still remain largely unknown. While recent advances show that stem cells can mimic embryogenesis1–9 to unravel hidden developmental mechanisms, a stem cell-based model of early human embryogenesis is lacking. Here, we use human extended pluripotent stem cells10to reconstitute early human development in 3-dimensions and recapitulate early embryo-like events. We first perform a systematic characterisation to reveal unique signalling requirements for building the human pre-implantation blastocyst. Further, we show that these in vitro stem cell-derived blastocyst-like structures are able to undertake spatiotemporal self-organisation to mimic peri-implantation remodelling in which a polarised rosette opens up the amniotic cavity within a developing disc. The hallmarks of human early development displayed by this stem cell-based in vitro model mimics features of embryonic day 3 to day 9/10 of natural development. Thus, this platform represents a tractable model system to contribute to the basic understanding of cellular and molecular mechanisms governing early embryonic events in humans and to provide valuable insights into the design of differentiation protocols for human stem cells in clinical applications.

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Concise Review; Effects of Antibiotics and Antimycotics on the Biological Properties of Human Pluripotent and Multipotent Stem Cells

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16999201203214425 ◽

2020 ◽

Vol 16 ◽

Author(s):

Maryam Farzaneh

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Culture Media ◽

Embryonic Stem ◽

Great Promise ◽

Human Stem Cells ◽

Multipotent Stem Cells ◽

The Impact

Abstract:: Human pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the remarkable potential to self-renew and develop into various cell lineages. Human mesenchymal stem cells (MSCs) or multipotent stem cells that are present in various organs can self-renew and differentiate into multiple mesenchymal lineages. Both human PSCs and MSCs hold great promise in cell-based therapies, disease modeling, drug discovery, and regenerative medicine. Human stem cells must be cultured under the optimal conditions to use them in transplantology. Therefore, researchers must ensure the sterility of human stem cell lines. Bacterial contamination is a common problem in laboratories and major precautions are required to detect the types of microorganisms, eliminate, and prevent contamination in cell cultures. Stem cell culture media usually contains antibiotics and antimycotics such as penicillin-streptomycin (pen-strep), gentamicin, and amphotericin B (AmB) to avoid bacterial, fungal, and yeast contaminants. Numerous publications recognized the serious effect of antibiotics and antimycotics on in vitro properties of human stem cells, including proliferation, differentiation, survival, and genetic instability. This review study aimed to understand the impact of routinely used antibiotics and antimycotics such as pen-strep, gentamicin, and AmB on viability, proliferation, and functional properties (differentiation and pluripotency) of human PSCs and MSCs.

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Nanobiosensing Platforms for Real-time and Non-Invasive Monitoring of Stem Cell Pluripotency and Differentiation

Sensors ◽

10.3390/s18092755 ◽

2018 ◽

Vol 18 (9) ◽

pp. 2755 ◽

Cited By ~ 10

Author(s):

Intan Rosalina Suhito ◽

Novi Angeline ◽

Sung-Sik Choo ◽

Ho Young Woo ◽

Taejong Paik ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Differentiation ◽

Electrochemical Sensing ◽

Non Invasive ◽

Differentiated Cells ◽

Present Information ◽

Stem Cell Pluripotency ◽

Technical Motivation ◽

Non Destructive

Breakthroughs in the biomedical and regenerative therapy fields have led to the influential ability of stem cells to differentiate into specific types of cells that enable the replacement of injured tissues/organs in the human body. Non-destructive identification of stem cell differentiation is highly necessary to avoid losses of differentiated cells, because most of the techniques generally used as confirmation tools for the successful differentiation of stem cells can result in valuable cells becoming irrecoverable. Regarding this issue, recent studies reported that both Raman spectroscopy and electrochemical sensing possess excellent characteristics for monitoring the behavior of stem cells, including differentiation. In this review, we focus on numerous studies that have investigated the detection of stem cell pluripotency and differentiation in non-invasive and non-destructive manner, mainly by using the Raman and electrochemical methods. Through this review, we present information that could provide scientific or technical motivation to employ or further develop these two techniques for stem cell research and its application.

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Histone Deacetylases in Neural Stem Cells and Induced Pluripotent Stem Cells

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/835968 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6 ◽

Cited By ~ 17

Author(s):

Guoqiang Sun ◽

Chelsea Fu ◽

Caroline Shen ◽

Yanhong Shi

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Neural Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Histone Deacetylases ◽

Molecular Mechanisms ◽

Self Renewal ◽

Stem Cell Pluripotency ◽

Induced Pluripotent

Stem cells have provided great hope for the treatment of a variety of human diseases. However, the molecular mechanisms underlying stem cell pluripotency, self-renewal, and differentiation remain to be unveiled. Epigenetic regulators, including histone deacetylases (HDACs), have been shown to coordinate with cell-intrinsic transcription factors and various signaling pathways to regulate stem cell pluripotency, self-renewal, and fate determination. This paper focuses on the role of HDACs in the proliferation and neuronal differentiation of neural stem cells and the application of HDAC inhibitors in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). It promises to be an active area of future research.

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Glycine cleavage system determines the fate of pluripotent stem cells via the regulation of senescence and epigenetic modifications

Life Science Alliance ◽

10.26508/lsa.201900413 ◽

2019 ◽

Vol 2 (5) ◽

pp. e201900413 ◽

Cited By ~ 1

Author(s):

Shengya Tian ◽

Junru Feng ◽

Yang Cao ◽

Shengqi Shen ◽

Yongping Cai ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Glycine Cleavage System ◽

Somatic Cell Reprogramming ◽

Stem Cell Pluripotency ◽

Glycine Cleavage ◽

Underlying Mechanisms ◽

Rate Limiting ◽

Pluripotency Genes

Metabolic remodelling has emerged as critical for stem cell pluripotency; however, the underlying mechanisms have yet to be fully elucidated. Here, we found that the glycine cleavage system (GCS) is highly activated to promote stem cell pluripotency and during somatic cell reprogramming. Mechanistically, we revealed that the expression of Gldc, a rate-limiting GCS enzyme regulated by Sox2 and Lin28A, facilitates this activation. We further found that the activated GCS catabolizes glycine to fuel H3K4me3 modification, thus promoting the expression of pluripotency genes. Moreover, the activated GCS helps to cleave excess glycine and prevents methylglyoxal accumulation, which stimulates senescence in stem cells and during reprogramming. Collectively, our results demonstrate a novel mechanism whereby GCS activation controls stem cell pluripotency by promoting H3K4me3 modification and preventing cellular senescence.

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CBP/p300 orthologs CBP2 and CBP3 play distinct roles in planarian stem cell function

10.1101/2020.09.08.287417 ◽

2020 ◽

Author(s):

Clara R. Stelman ◽

Britessia M. Smith ◽

Bidushi Chandra ◽

Rachel H. Roberts-Galbraith

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Cell Biology ◽

Chromatin Modification ◽

Whole Body ◽

Pluripotent State ◽

Stem Cell Maintenance ◽

Cell Maintenance

AbstractChromatin modifications function as critical regulators of gene expression and cellular identity, especially in the regulation and maintenance of the pluripotent state. However, many studies of chromatin modification in stem cells—and pluripotent stem cells in particular—are performed in mammalian stem cell culture, an in vitro condition mimicking a very transient state during mammalian development. Thus, new models for study of pluripotent stem cells in vivo could be helpful for understanding the roles of chromatin modification, for confirming prior in vitro studies, and for exploring evolution of the pluripotent state. The freshwater flatworm, Schmidtea mediterranea, is an excellent model for studying adult pluripotent stem cells, particularly in the context of robust, whole-body regeneration. To identify chromatin modifying and remodeling enzymes critical for planarian regeneration and stem cell maintenance, we took a candidate approach and screened planarian homologs of 26 genes known to regulate chromatin biology in other organisms. Through our study, we identified six genes with novel functions in planarian homeostasis, regeneration, and behavior. We also identified in our list five planarian homologs of the mammalian CREB-Binding Protein (CBP) family of histone acetyltransferases, representing an expansion of this family in planarians. We find that two planarian CBP family members are required for planarian survival, with knockdown of Smed-CBP2 and Smed-CBP3 causing distinct defects in stem cell maintenance or function. Loss of CBP2 causes a quick, dramatic loss of stem cells, while knockdown of CBP3 more narrowly affects stem cells, preferentially decreasing markers of neural progenitors. We propose that the division of labor among a diversified CBP family in planarians presents an opportunity to dissect specific functions of a broadly important histone acetyltransferase family in stem cell biology.

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Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

Stem Cells International ◽

10.1155/2014/409021 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 4

Author(s):

Jitong Guo ◽

Baojiang Wu ◽

Shuyu Li ◽

Siqin Bao ◽

Lixia Zhao ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Embryonic Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Germline Transmission ◽

Stem Cell Pluripotency ◽

Induced Pluripotent

Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cell embryos with embryonic stem cells. The fully agouti colored chimeras were generated from both injection and coculture of 8-cell embryos with embryonic stem cells. Additionally, microsatellite DNA screening showed that the fully agouti colored chimeras were fully embryonic stem cell derived mice. Unlike embryonic stem cells, the mouse chimeras were only generated from injection of 8-cell embryos with induced pluripotent stem cells and none of these showed germline transmission. The results indicated that injection of 8-cell embryos is the most efficient method for assessing stem cell pluripotency and generating induced pluripotent stem cell chimeras, embryonic stem cell chimeras with germline transmission, and fully mouse embryonic stem cell derived mice.

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Selective Ablation of Cochlear Hair Cells Promotes Engraftment of Human Embryonic Stem Cells-derived Progenitors in The Mouse organ of Corti

10.21203/rs.3.rs-132720/v1 ◽

2020 ◽

Author(s):

Hiroki Takeda ◽

Anna Dondzillo ◽

Jessica A. Randall ◽

Samuel P. Gubbels

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Inner Ear ◽

Hair Cells ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Organ Of Corti ◽

Human Stem Cells ◽

Cochlear Hair Cells ◽

Mouse Organ

Abstract Backgroud: Hearing loss affects 25% of the population at ages 60–69 years. Loss of the hair cells of the inner ear commonly underlies deafness and once lost this cell type cannot spontaneously regenerate in higher vertebrates. As a result there is a need for the development of regenerative strategies to replace hair cells once lost. Stem cell-based therapies are one such strategy and offer promise for cell replacement in a variety of tissues. A number of investigators have previously demonstrated successful implantation, and certain level of regeneration of hair and supporting cells in both avian and mammalian models using rodent pluripotent stem cells. However, the ability of human stem cells to engraft and generate differentiated cell types in the inner ear is not well understood. Methods: We differentiate human pluripotent stem cells to the pre-placodal stage in vitro then transplant them into the mouse cochlea after selective and complete lesioning of the endogenous population of hair cells. Results: We demonstrate that hair cell ablation prior to transplantation leads to increased engraftment in the auditory sensory epithelium, the organ of Corti, as well as differentiation of transplanted cells into hair and supporting cell immunophenotypes. Conclusion: We have demonstrated the feasibility of human stem cell engraftment into an ablated mouse organ of Corti.

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Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells

Cells ◽

10.3390/cells10102622 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2622

Author(s):

Preeti Ashok ◽

Emmanuel S. Tzanakakis

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Proteomic Analysis ◽

Protein Composition ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Cardiac Diseases ◽

Cardiogenic Differentiation

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.

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