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Developmental and Reproductive Toxicology pp 279–312Cite as

High-Throughput Screens for Embryonic Stem Cells: Stress-Forced Potency-Stemness Loss Enables Toxicological Assays

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Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Embryonic stem cells (ESCs) respond to the majority of toxicological stresses by growing more slowly, and a subpopulation of cells loses all of a subset of nuclear potency/stemness factors. Slowed growth and potency factor loss occur despite culture of ESCs under conditions that favor proliferation and potency maintenance. Stemness/potency factor loss enables the stem cells to differentiate to create essential first lineage functions. Increase in the fraction of stem cells with potency loss and differentiation increase, in response to diminished population growth, is called compensatory differentiation since increased differentiation compensates for diminished population growth.

To take advantage of compensatory differentiation where the potency marker Rex1 undergoes permanent stress-induced loss (Slater et al., Stem Cells Dev 23:3049–3064, 2014), a high throughput screen (HTS1 ESC) has been created in ESCs. Using low-stress lentivirus infection and low-stress FACS isolation, ESC lines were created that are transgenic for gene constructs that use either or both Rex1 potency factor promoter to drive red fluorescence protein (Rex1-RFP) and/or Oct4 potency factor promoter to drive green fluorescent (Oct4-GFP) (Li et al., Stem Cells Dev 25:320–328, 2016). Four independent assays showed that control hyperosmotic stress caused Rex1-RFP to decrease. These assays were immunofluorescence, microplate reader, flow cytometry, and Western blot. In addition, Hoechst was used to assay cell number at the start and end of treatment in order to quantitate growth rate. Since potency factor loss enables differentiation increase then stress-forced differentiation should compensate for diminished stem cell population size. Stress dose-dependent induction of decreased Rex1-RFP was similar to, and corroborated, by dose-dependent loss of endogenous Rex1 protein. This assay shows promise in reporting toxicological stresses.

Potency loss using Rex1-RFP has been quantitated and at a nonmorbid stress dose approaches a 20 % increase over unstressed ESCs. The quantitation of the gain of differentiation in subpopulation size is underway [Li et al., Development (manuscript in preparation)], and hypothetically is within the 20 % of cells with complete (e.g., to the level of non-transgenic parental ESCs) Rex1-RFP loss. Four days after fertilization the ESC lineage arises in the blastocyst stage embryo. At the same time the distinct placental trophoblast stem cell lineage (TSC) also arise and these cells undergo a 50 % induction of giant cell differentiation as measured by morphology, and gain of first lineage differentiated markers. For both ESC and TSC, stress-forced potency loss and differentiation increase occurs despite culture conditions that should maintain proliferation and potency. Thus these assays measure stem cells depletion due to stress forced growth and potency decrease, and differentiation increase. Production of a TSC HTS is important as this stress-forced differentiation is larger than ESC effects and is linked to events that are known to lead to miscarriage.

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Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI, 48201, USA

    Quanwen Li, Erica Louden & Daniel A. Rappolee

  2. Department of Physiology, Reproductive Sciences Program, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Daniel A. Rappolee

  3. Department of Obstetrics/Gynecology, Reproductive Sciences Program, University School of Medicine, Detroit, MI, 48201, USA

    Elizabeth E. Puscheck

  4. Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Daniel A. Rappolee

  5. Institutes for Environmental Health Science, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Daniel A. Rappolee

  6. Department of Biology, University of Windsor, Windsor, ON, Canada, N9B 3P4

    Daniel A. Rappolee

  7. Founder of the S Corp; Reproductive Stress (3M); Markers, Mechanisms, and Management, Detroit, MI, USA

    Elizabeth E. Puscheck & Daniel A. Rappolee

  8. Department of Obstetrics and Gynecology, Department of Physiology, C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI, 48201, USA

    Yu Yang

Authors
  1. Quanwen Li
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  2. Yu Yang
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  3. Erica Louden
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  4. Elizabeth E. Puscheck
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  5. Daniel A. Rappolee
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Corresponding author

Correspondence to Daniel A. Rappolee .

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Editors and Affiliations

  1. MPI Research, Mattawan, Michigan, USA

    Ali S. Faqi

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Li, Q., Yang, Y., Louden, E., Puscheck, E.E., Rappolee, D.A. (2016). High-Throughput Screens for Embryonic Stem Cells: Stress-Forced Potency-Stemness Loss Enables Toxicological Assays. In: Faqi, A. (eds) Developmental and Reproductive Toxicology. Methods in Pharmacology and Toxicology. Humana Press, New York, NY. https://doi.org/10.1007/7653_2016_66

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  • DOI: https://doi.org/10.1007/7653_2016_66

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7206-7

  • Online ISBN: 978-1-4939-7208-1

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