Publications by authors named "Amander T Clark"

89 Publications

Generation of three human induced pluripotent stem cell sublines (UCLAi004-A, UCLAi004-B, and UCLAi004-C) for reproductive science research.

Stem Cell Res 2021 Jul 24;54:102446. Epub 2021 Jun 24.

Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA. Electronic address:

Three induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal human dermal fibroblasts (HDFs) derived from a human skin punch biopsy. The biopsy was donated from a woman with known infertility due to ovarian failure. The hiPSC sublines were created using Sendai virus vectors and were positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was verified using PluriTest analysis and in vitro differentiation using Taqman Real-Time PCR assays for somatic lineage markers. This participant's monozygotic twin sister also donated a skin-punch biopsy, whose resulting hiPSC lines were published previously as a resource.
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http://dx.doi.org/10.1016/j.scr.2021.102446DOI Listing
July 2021

Generation of three human induced pluripotent stem cell sublines (UCLAi005-A, UCLAi005-B and UCLAi005-C) for reproductive science research.

Stem Cell Res 2021 Jul 8;54:102409. Epub 2021 Jun 8.

Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA. Electronic address:

We generated three human induced pluripotent stem cell (hiPSC) sublines from human dermal fibroblasts (HDF) (MZT05) generated from a skin biopsy donated from a previously fertile woman. The skin biopsy was broadly consented for generating hiPSC lines for biomedical research, including unique consent specifically for studying human fertility, infertility and germ cell differentiation. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal. Pluripotency was further verified using PluriTest analysis and in vitro differentiation was tested using Taqman Real-Time PCR assays. These sublines serve as controls for hiPSC research projects aimed at understanding the cell and molecular regulation of female fertility.
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http://dx.doi.org/10.1016/j.scr.2021.102409DOI Listing
July 2021

Human embryo research, stem cell-derived embryo models and in vitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

Stem Cell Reports 2021 Jun 27;16(6):1416-1424. Epub 2021 May 27.

Gairdner Foundation, Toronto, Ontario, Canada. Electronic address:

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190666PMC
June 2021

Postpubertal spermatogonial stem cell transplantation restores functional sperm production in rhesus monkeys irradiated before and after puberty.

Andrology 2021 May 6. Epub 2021 May 6.

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario.

Objectives: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period.

Materials And Methods: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses.

Results: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells.

Discussion And Conclusion: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.
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http://dx.doi.org/10.1111/andr.13033DOI Listing
May 2021

Modelling human blastocysts by reprogramming fibroblasts into iBlastoids.

Nature 2021 Mar 17;591(7851):627-632. Epub 2021 Mar 17.

Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.
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http://dx.doi.org/10.1038/s41586-021-03372-yDOI Listing
March 2021

Mammalian primordial germ cell specification.

Development 2021 Mar 15;148(6). Epub 2021 Mar 15.

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA

The peri-implantation window of mammalian development is the crucial window for primordial germ cell (PGC) specification. Whereas pre-implantation dynamics are relatively conserved between species, the implantation window marks a stage of developmental divergence between key model organisms, and thus potential variance in the cell and molecular mechanisms for PGC specification. In humans, PGC specification is very difficult to study To address this, the combined use of human and nonhuman primate embryos, and stem cell-based embryo models are essential for determining the origin of PGCs, as are comparative analyses to the equivalent stages of mouse development. Understanding the origin of PGCs in the peri-implantation embryo is crucial not only for accurate modeling of this essential process using stem cells, but also in determining the role of global epigenetic reprogramming upon which sex-specific differentiation into gametes relies.
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http://dx.doi.org/10.1242/dev.189217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990907PMC
March 2021

Generation of six human induced pluripotent stem cell sublines (MZT01E, MZT01F, MZT01N and MZT02D, MZT02G and MZT02H) for reproductive science research.

Stem Cell Res 2021 03 27;51:102204. Epub 2021 Jan 27.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA.

Six human induced pluripotent stem cell sublines (hiPSCs) were generated from human dermal fibroblasts (HDFs) derived from skin biopsies donated from monozygotic twin women wherein one woman had proven fertility and her sister was infertile due to ovarian failure. Three hiPSC sublines were created from each twin's HDFs. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was further verified using PluriTest. We show here that the hiPSC lines created from the twins are equivalent in measures of pluripotency and self-renewal, despite their differential diagnosis.
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http://dx.doi.org/10.1016/j.scr.2021.102204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043044PMC
March 2021

Single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment.

Cell Stem Cell 2021 04 15;28(4):764-778.e4. Epub 2021 Jan 15.

Howard Hughes Medical Institute, Department of Oncological Sciences and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. Electronic address:

Human testis development in prenatal life involves complex changes in germline and somatic cell identity. To better understand, we profiled and analyzed ∼32,500 single-cell transcriptomes of testicular cells from embryonic, fetal, and infant stages. Our data show that at 6-7 weeks postfertilization, as the testicular cords are established, the Sertoli and interstitial cells originate from a common heterogeneous progenitor pool, which then resolves into fetal Sertoli cells (expressing tube-forming genes) or interstitial cells (including Leydig-lineage cells expressing steroidogenesis genes). Almost 10 weeks later, beginning at 14-16 weeks postfertilization, the male primordial germ cells exit mitosis, downregulate pluripotent transcription factors, and transition into cells that strongly resemble the state 0 spermatogonia originally defined in the infant and adult testes. Therefore, we called these fetal spermatogonia "state f0." Overall, we reveal multiple insights into the coordinated and temporal development of the embryonic, fetal, and postnatal male germline together with the somatic niche.
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http://dx.doi.org/10.1016/j.stem.2020.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026516PMC
April 2021

Reprogramming roadmap reveals route to human induced trophoblast stem cells.

Nature 2020 10 16;586(7827):101-107. Epub 2020 Sep 16.

Monash Bioinformatics Platform, Monash University, Melbourne, Victoria, Australia.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.
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http://dx.doi.org/10.1038/s41586-020-2734-6DOI Listing
October 2020

An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

Stem Cell Reports 2020 03 13;14(3):433-446. Epub 2020 Feb 13.

Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

The development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.
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http://dx.doi.org/10.1016/j.stemcr.2020.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066331PMC
March 2020

Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Cell Rep 2019 12;29(11):3488-3505.e9

UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; Eli and Edythe Broad Stem Cell Research Center, UCLA, Los Angeles, CA 90095, USA; UCLA Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA. Electronic address:

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.
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http://dx.doi.org/10.1016/j.celrep.2019.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927560PMC
December 2019

Standing on the shoulders of giants: The changing landscape of pluripotent stem cells in research.

Authors:
Amander T Clark

Anat Rec (Hoboken) 2020 10 21;303(10):2597-2602. Epub 2019 Nov 21.

Department of Molecular Cell and Developmental Biology, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles, California.

Stem cells have the remarkable property of self-renewal and differentiation. These two fundamental aspects have excited scientists and clinicians for decades. Stem cells are defined by their potency, with pluripotency being the most permissive and unipotency being the most restricted. In mammals, pluripotency represents cell types found in the preimplantation and early postimplantation embryo. However, these pluripotent cells are not stem cells per se, because they do not meet the criteria of self-renewal. Therefore, pluripotent stem cells are exclusively in vitro cell types that have provided scientists and clinicians with unprecedented power to study the fundamental cell and molecular properties of pluripotency, as well as providing a window into cellular differentiation and a source of cells for regenerative medicine including cell types that could be used to regenerate the kidney.
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http://dx.doi.org/10.1002/ar.24304DOI Listing
October 2020

Generation of three human induced pluripotent stem cell sublines (MZT04D, MZT04J, MZT04C) for reproductive science research.

Stem Cell Res 2019 10 16;40:101576. Epub 2019 Sep 16.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA. Electronic address:

We generated three human induced pluripotent stem cell (hiPSC) sublines from human dermal fibroblasts (HDFs) (MZT04) generated from a skin biopsy donated from a previously fertile woman. The skin biopsy was broadly consented for generating hiPSC lines for biomedical research, including unique consent specifically for studying human fertility, infertility and germ cells. hiPSCs were reprogrammed using Sendai virus vectors and were subsequently positive for markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA-4. Pluripotency was further verified using teratomas and PluriTest. These sublines serve as controls for hiPSC research projects aimed at understanding the cell and molecular regulation of female fertility and infertility.
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http://dx.doi.org/10.1016/j.scr.2019.101576DOI Listing
October 2019

The TFAP2C-Regulated OCT4 Naive Enhancer Is Involved in Human Germline Formation.

Cell Rep 2018 12;25(13):3591-3602.e5

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA, USA. Electronic address:

Human primordial germ cells (hPGCs) are the first embryonic progenitors in the germ cell lineage, yet the molecular mechanisms required for hPGC formation are not well characterized. To identify regulatory regions in hPGC development, we used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to systematically characterize regions of open chromatin in hPGCs and hPGC-like cells (hPGCLCs) differentiated from human embryonic stem cells (hESCs). We discovered regions of open chromatin unique to hPGCs and hPGCLCs that significantly overlap with TFAP2C-bound enhancers identified in the naive ground state of pluripotency. Using CRISPR/Cas9, we show that deleting the TFAP2C-bound naive enhancer at the OCT4 locus (also called POU5F1) results in impaired OCT4 expression and a negative effect on hPGCLC identity.
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http://dx.doi.org/10.1016/j.celrep.2018.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342560PMC
December 2018

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

Nat Commun 2018 12 17;9(1):5339. Epub 2018 Dec 17.

Department of Molecular, Cell and Developmental Biology, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, 90095, USA.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia.
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http://dx.doi.org/10.1038/s41467-018-07740-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297357PMC
December 2018

Restoring Fertility with Human Induced Pluripotent Stem Cells: Are We There Yet?

Cell Stem Cell 2018 12;23(6):777-779

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA. Electronic address:

Overcoming infertility with assisted reproduction requires high-quality eggs and sperm. For those young women who no longer make functional eggs, the hope of conceiving a biological child just got one step closer with a recent publication in Science from Yamashiro et al. (2018).
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http://dx.doi.org/10.1016/j.stem.2018.11.003DOI Listing
December 2018

Editorial.

Stem Cell Res 2018 05 13;29:179. Epub 2018 Apr 13.

Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, United States. Electronic address:

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http://dx.doi.org/10.1016/j.scr.2018.04.006DOI Listing
May 2018

TFAP2C regulates transcription in human naive pluripotency by opening enhancers.

Nat Cell Biol 2018 05 25;20(5):553-564. Epub 2018 Apr 25.

Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA.

Naive and primed pluripotent human embryonic stem cells bear transcriptional similarity to pre- and post-implantation epiblast and thus constitute a developmental model for understanding the pluripotent stages in human embryo development. To identify new transcription factors that differentially regulate the unique pluripotent stages, we mapped open chromatin using ATAC-seq and found enrichment of the activator protein-2 (AP2) transcription factor binding motif at naive-specific open chromatin. We determined that the AP2 family member TFAP2C is upregulated during primed to naive reversion and becomes widespread at naive-specific enhancers. TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. Additionally, we identify a previously undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for TFAP2C binding. Taken together, TFAP2C establishes and maintains naive human pluripotency and regulates OCT4 expression by mechanisms that are distinct from mouse.
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http://dx.doi.org/10.1038/s41556-018-0089-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926822PMC
May 2018

Mitochondrial DNA selection in human germ cells.

Nat Cell Biol 2018 02;20(2):118-120

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, CA, USA.

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http://dx.doi.org/10.1038/s41556-017-0029-4DOI Listing
February 2018

PRDM14 is expressed in germ cell tumors with constitutive overexpression altering human germline differentiation and proliferation.

Stem Cell Res 2018 03 4;27:46-56. Epub 2018 Jan 4.

Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Los Angeles, CA 90095, USA; University of California Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Germ cell tumors (GCTs) are a heterogeneous group of tumors occurring in gonadal and extragonadal locations. GCTs are hypothesized to arise from primordial germ cells (PGCs), which fail to differentiate. One recently identified susceptibility loci for human GCT is PR (PRDI-BF1 and RIZ) domain proteins 14 (PRDM14). PRDM14 is expressed in early primate PGCs and is repressed as PGCs differentiate. To examine PRDM14 in human GCTs we profiled human GCT cell lines and patient samples and discovered that PRDM14 is expressed in embryonal carcinoma cell lines, embryonal carcinomas, seminomas, intracranial germinomas and yolk sac tumors, but is not expressed in teratomas. To model constitutive overexpression in human PGCs, we generated PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs) and discovered that elevated expression of PRDM14 does not block early PGC formation. Instead, we show that elevated PRDM14 in PGCLCs causes proliferation and differentiation defects in the germline.
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http://dx.doi.org/10.1016/j.scr.2017.12.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858915PMC
March 2018

TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency.

Stem Cell Reports 2018 01 28;10(1):243-256. Epub 2017 Dec 28.

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not.
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http://dx.doi.org/10.1016/j.stemcr.2017.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768987PMC
January 2018

Germline competency of human embryonic stem cells depends on eomesodermin.

Biol Reprod 2017 01;97(6):850-861

Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, California, USA.

In humans, germline competency and the specification of primordial germ cells (PGCs) are thought to occur in a restricted developmental window during early embryogenesis. Despite the importance of specifying the appropriate number of PGCs for human reproduction, the molecular mechanisms governing PGC formation remain largely unexplored. Here, we compared PGC-like cell (PGCLC) differentiation from 18 independently derived human embryonic stem cell (hESC) lines, and discovered that the expression of primitive streak genes were positively associated with hESC germline competency. Furthermore, we show that chemical inhibition of TGFβ and WNT signaling, which are required for primitive streak formation and CRISPR/Cas9 deletion of Eomesodermin (EOMES), significantly impacts PGCLC differentiation from hESCs. Taken together, our results suggest that human PGC formation involves signaling and transcriptional programs associated with somatic germ layer induction and expression of EOMES.
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http://dx.doi.org/10.1093/biolre/iox138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803789PMC
January 2017

An integration-free, virus-free rhesus macaque induced pluripotent stem cell line (riPSC90) from embryonic fibroblasts.

Stem Cell Res 2017 05 15;21:5-8. Epub 2017 Mar 15.

Department of Molecular, Cell and Developmental Biology, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

The rhesus macaque induced pluripotent stem cell (riPSC) line, UCLAi090-A (riPSC90), was generated from rhesus embryonic fibroblast (REF) cells called REF90. REF90 cells and the riPSC90 line were authenticated by short tandem repeat analysis and had a normal male (42, XY) karyotype. The riPSC90 line expressed markers of self-renewal including OCT4, NANOG, TRA-1-81 and SSEA4, and generated teratomas after transplantation into immunocompromised mice. riPSC90 could be used in parallel with riPSC89, which was derived from REFs cultured from a different rhesus macaque embryo (Sosa et al. 2016).
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http://dx.doi.org/10.1016/j.scr.2017.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499526PMC
May 2017

Primate Primordial Germ Cells Acquire Transplantation Potential by Carnegie Stage 23.

Stem Cell Reports 2017 07 1;9(1):329-341. Epub 2017 Jun 1.

Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Primordial germ cells (PGCs) are the earliest embryonic progenitors in the germline. Correct formation of PGCs is critical to reproductive health as an adult. Recent work has shown that primate PGCs can be differentiated from pluripotent stem cells; however, a bioassay that supports their identity as transplantable germ cells has not been reported. Here, we adopted a xenotransplantation assay by transplanting single-cell suspensions of human and nonhuman primate embryonic Macaca mulatta (rhesus macaque) testes containing PGCs into the seminiferous tubules of adult busulfan-treated nude mice. We discovered that both human and nonhuman primate embryonic testis are xenotransplantable, generating colonies while not generating tumors. Taken together, this work provides two critical references (molecular and functional) for defining transplantable primate PGCs. These results provide a blueprint for differentiating pluripotent stem cells to transplantable PGC-like cells in a species that is amenable to transplantation and fertility studies.
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http://dx.doi.org/10.1016/j.stemcr.2017.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511048PMC
July 2017

Modeling human infertility with pluripotent stem cells.

Stem Cell Res 2017 05 13;21:187-192. Epub 2017 Apr 13.

Department of Molecular, Cell and Developmental Biology, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Los Angeles, CA 90095, USA; University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification in order to understand human fertility.
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http://dx.doi.org/10.1016/j.scr.2017.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498156PMC
May 2017

Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation.

Cell 2017 Jan 12;168(1-2):210-223.e11. Epub 2017 Jan 12.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development.
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http://dx.doi.org/10.1016/j.cell.2016.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321559PMC
January 2017

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

Stem Cells 2017 03 19;35(3):626-640. Epub 2017 Jan 19.

Clayton and Parkville, CSIRO Manufacturing, Victoria, Australia.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.
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http://dx.doi.org/10.1002/stem.2558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412944PMC
March 2017

Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation.

Cell Stem Cell 2017 01 15;20(1):87-101. Epub 2016 Dec 15.

Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Bioinformatics Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (X) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XX intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets X abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.
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http://dx.doi.org/10.1016/j.stem.2016.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5218861PMC
January 2017