Fate of iPSCs Derived from Azoospermic and Fertile Men following Xenotransplantation to Murine Seminiferous Tubules

SummaryHistorically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC) lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF) regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs) that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status

Characterization of the human ESC transcriptome by hybrid sequencing

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, fulllength mRNA isoforms are not captured. On the other hand, thirdgeneration sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency- associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete

Differenzierung von induzierten pluripotenten Stammzellen in murinen Samenkanälchen

Die Unzulänglichkeit von humanen Keimzellen während der Embryonalentwicklung haben Studien über dieselbigen bisher vor eine Herausforderung gestellt. Einige Veröffentlichungen haben zwar gezeigt, dass humane Keimzellen aus pluripotenten Stammzellen in vitro differenziert werden können, allerdings war dies in vivo bisher nicht erfolgreich. In dieser Arbeit haben wir in einleitenden Untersuchungen die Genexpression von kürzlich neu entdeckten Genen während der Zellreprogrammierung auf Einzell-Ebene erforscht. Weiterführend haben wir getestet, ob mRNA Reprogrammierung in Kombination mit Xenotransplantation eine durchführbare Strategie für die Untersuchung der humanen Keimzellentwicklung aus induzierten pluripotenten Stammzellen (iPSCs) ist. Wir produzierten integrationsfreie iPSCs mit Hilfe der mRNA-basierten Reprogrammierung entweder mit OCT3/4, SOX2, KLF4 und cMYC (OSKM) oder in Kombination mit einer spezifischen Keimzellmarker-mRNA, VASA (OSKMV). Alle iPSC Linien erfüllten die Kriterien für ihren pluripotenten Phenotyp. Desweiteren haben globale Genexpressionsanalysen zwischen OSKM und OSKMV hergestellten iPSCs nicht unterscheiden können. Nur geringe Unterschiede wurden hinsichtlich der Genexpression von keimzellspezifischen Genen, epigenetische Profilen und in vitro Studien entdeckt. Demgegenüber zeigten beide Linien (OSKM und OSKMV) signikante Unterschiede hinsichtlich ihres Differenzierungspotentials, als sie direkt in die Samenkanälchen von immungeschwächten Mäusen transplantiert wurden, deren endogene Keimzellen zerstört wurden. Transplantation der undifferenzierten Stammzelllinien resultierte in morphologisch und immunohistochemisch erkennbare Keimzellen in vivo, insbesondere im Fall von OSKMV Zellen. Dabei ist hervorzuheben, dass OSKMV Zellen, die außerhalb der Samenkanälchen verblieben waren, im Gegensatz zu OSKM Zellen, keine Tumore bildeten. Letztere proliferierten unkontrolliert und formten Tumore. Die Resultate zeigen, dass mRNA Reprogrammierung in Kombination mit Zelltransplantation eine brauchbare Strategie hinsichtlich genetischer Analysen der humanen Keimzellentwicklung ist. Zusätzlich können sie hilfreich für Studien sein, die die Wiederherstellung der Fruchtbarkeit von Männern untersuchen.Studies of human germ cell development are limited in large part due to inaccessibility of germ cells during development. Moreover, although several studies have reported differentiation of mouse and human germ cells from pluripotent stem cells in vitro, differentiation of human germ cells from pluripotent stem cells in vivo has not been reported. Here, we investigate in preliminary studies dynamic gene expression changes of recently discovered novel genes during nuclear cell reprogramming on a single cell level. We then test whether mRNA reprogramming in combination with xeno-transplantation may provide a viable system to probe the genetics of human germ cell development via use of induced pluripotent stem (iPS) cells. For this purpose, we derived integration-free iPS cells via mRNA-based reprogramming with OCT3/4, SOX2, KLF4 and cMYC alone (OSKM) or in combination with the germ cell specific mRNA, VASA (OSKMV). All iPS cell lines met classic criteria of pluripotency. Moreover, global gene expression proling did not distinguish between undifferentiated OSKM and OSKMV iPS cells and only subtle differences were observed in expression of germ cell specific genes, epigenetic profiles and in vitro differentiation studies. In contrast, transplantation of undifferentiated iPS cells directly into the seminiferous tubules of germ cell-depleted immunodecient mice revealed divergent fates of iPS cells produced with different factors. Transplantation resulted in morphologically and immunohistochemically recognizable germ cells in vivo, particularly in the case of OSKMV cells. Significantly, OSKMV cells also did not form tumors while OSKM cells that remained outside the seminiferous tubule proliferated extensively and formed tumors. Results indicate that mRNA reprogramming in combination with transplantation is a viable strategy for genetic analysis of human germ cell development and may inform studies of fertility restoration in men

Single cell expression analysis of primate-specific retroviruses-derived HPAT lincRNAs in viable human blastocysts identifies embryonic cells co-expressing genetic markers of multiple lineages

Chromosome instability and aneuploidies occur very frequently in human embryos, impairing proper embryogenesis and leading to cell cycle arrest, loss of cell viability, and developmental failures in 50–80% of cleavage-stage embryos. This high frequency of cellular extinction events represents a significant experimental obstacle challenging analyses of individual cells isolated from human preimplantation embryos. We carried out single cell expression profiling of 241 individual cells recovered from 32 human embryos during the early and late stages of viable human blastocyst (VHB) differentiation. Classification of embryonic cells was performed solely based on expression patterns of human pluripotency-associated transcripts (HPAT), which represent a family of primate-specific transposable element-derived lincRNAs highly expressed in human embryonic stem cells and regulating nuclear reprogramming and pluripotency induction. We then validated our findings by analyzing transcriptomes of 1,708 individual cells recovered from more than 100 human embryos and 259 mouse cells from more than 40 mouse embryos at different stages of preimplantation embryogenesis. HPAT's expression-guided spatiotemporal reconstruction of human embryonic development inferred from single-cell expression analysis of VHB differentiation enabled identification of telomerase-positive embryonic cells co-expressing key pluripotency regulatory genes and genetic markers of three major lineages. Follow-up validation analyses confirmed the emergence in human embryos prior to lineage segregation of telomerase-positive cells co-expressing genetic markers of multiple lineages. Observations reported in this contribution support the hypothesis of a developmental pathway of creation embryonic lineages and extraembryonic tissues from telomerase-positive pre-lineage cells manifesting multi-lineage precursor phenotype

YAP Induces Human Naive Pluripotency

The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP−/− cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology

Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes

Generation and characterization of transgene-free human induced pluripotent stem cells and conversion to putative clinical-grade status

Abstract Introduction The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. Methods In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. Results We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. Conclusion For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics

<i>In vitro</i> and <i>in vivo</i> differentiation of GMP-transitioned RiPSC lines.

<p>(<b>A</b>) Directed <i>in vitro</i> differentiation of three GMP-transitioned RiPSC lines into three germ layers followed by FACS analysis (endoderm) and immunocytochemistry (ectoderm, mesoderm). Scale bar = 150 μm. (<b>B</b>) Comparison of differentiation potential into neuroectoderm (PAX6) and mesoderm (DESMIN) between non-GMP (research-grade) and GMP-grade lines (BJ, HUF1). Comparisons are based on counting analysis of cells that were positive for the specific differentiation markers. N = 200–300. (<b>C</b>) Hematoxylin and eosin staining of teratomas derived from GMP-transitioned lines showing ectoderm (neural rosettes, epidermis), mesoderm (cartilage) and endoderm (gut-like endothelium). Scale bar = 200 μm.</p