Generation of induced pluripotent stem cells from Bornean orangutans
Ontology highlight
ABSTRACT: Bornean orangutans (Pongo pygmaeus) are an endangered non-human primate species. Induced pluripotent stem cells (iPSCs) offer a promising avenue for preserving genetic resources and studying evolutionary processes. In this study, we successfully generate Bornean orangutan iPSCs (o-iPSCs) from peripheral blood mononuclear cells using Sendai virus-mediated reprogramming. Furthermore, we transform primed o-iPSCs into a naïve pluripotent state using a novel 4i/L/A culture system. The resulting naïve o-iPSCs exhibit key features similar to human naïve stem cells, including upregulation of KLF17, DNMT3L, and DPPA3/5. We also observe significant activation of the WNT signaling pathway and X chromosome reactivation in the 4i/L/A o-iPSCs. Transcriptome analysis confirmed their resemblance to human naïve embryonic stem cells. Our findings contribute to the molecular understanding of naïve o-iPSCs and provide a foundation for orangutan conservation using advanced technologies.
Project description:Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.
Project description:The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) and plays an important role in survival and maintenance of primordial germ cells (PGCs) and the development of the male germline in zebrafish and mice. It was shown to be expressed in human pluripotent stem cells (PSCs), PGCs, and spermatogonia, but little is known about its specific role in pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to analyse if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines with biallelic loss of function mutations and analysed their potential to differentiate towards PGCLCs and their gene expression on RNA and protein level via bulk RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation nor trilineage differentiation potential, but yielded reduced numbers o PGCLCs compared to their parental iPSCs. RNAseq analysis in PGCLCs showed significantly reduced expression of genes associated with cellular developmental processes and cell differentiation in knockout cells, including known markers for PGCs (NANOS3, SOX17, PRDM1, EPCAM) and naïve pluripotency (TFCP2L, DNMT3L).
Project description:Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naïve pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naïve pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naïve human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naïve pluripotency from other mammal species. In addition, the derivation of rhesus monkey naïve iPSCs also provides a valuable cell source for use in preclinical research and disease modeling. mRNA expression analysis of 4 rhesus monkey naive iPSC lines and 2 primed iPSC lines were examed.
Project description:The northern white rhinoceros (NWR) is probably the earth’s most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.
Project description:Orangutans are an endangered species whose natural habitats are restricted to the Southeast Asian islands of Borneo and Sumatra. For potential species conservation and functional genomics studies, we derived induced pluripotent stem cells (iPSCs) from cryopreserved skin fibroblasts obtained from captive orangutans. We report the gene expression profiles of iPSCs and skin fibroblasts derived from orangtuans.
Project description:Conventional embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) derived from primates resemble mouse epiblast stem cells, raising an intriguing question regarding whether the naïve pluripotent state resembling mouse embryonic stem cells (mESCs) exists in primates and how to capture it in vitro. Here we identified several specific signaling modulators that are sufficient to generate rhesus monkey fibroblast-derived iPSCs with the features of naïve pluripotency in terms of growth properties, gene expression profiles, self-renewal signaling, X-reactivation and the potential to generate cross-species chimeric embryos. Interestingly, together with recent reports of naïve human pluripotent stem cells, our findings suggest several conserved signaling pathways shared with rodents and specific to primates, providing significant insights for acquiring naïve pluripotency from other mammal species. In addition, the derivation of rhesus monkey naïve iPSCs also provides a valuable cell source for use in preclinical research and disease modeling.
Project description:Comparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically even impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that Sendai virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to generate primate iPSCs non-invasively from urinary samples.
Project description:Induced pluripotent stem cells (iPSCs) can provide biological resource for functional and conservation research for various species. However, the understanding of species variations of mammalian iPSCs is still limited. Here, we report the first generation of iPSCs from the endangered species Grevy's zebra (Equus grevyi; gz-iPSCs). We reprogram primary fibroblasts with human reprogramming transcription factors, OCT3/4, SOX2, KLF4, and c-MYC, with the retroviral method and confirmed the pluripotency and differentiation potential. In light of RNA sequencing analysis, generated gz-iPSCs robustly express genes associated with pluripotency and reprogramming processes, including epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT-MET). Comparative transcriptomics with other species reveals patterns of gene expressions among mammalian PSCs and detects evolutionary conservation of pluripotency-associated genes and plausible importance of translation process. This work will aid in providing biological resource for this endangered species and enables new insight into the evolution of the mammalian PSCs.
Project description:Orangutans are an endangered species whose natural habitats are restricted to the Southeast Asian islands of Borneo and Sumatra. For potential species conservation and functional genomics studies, we derived induced pluripotent stem cells (iPSCs) from cryopreserved skin fibroblasts obtained from captive orangutans. We report the gene expression profiles of iPSCs and skin fibroblasts derived from orangtuans. The overall goal was to evaluate gene expression biomarkers of pluripotency in iPSCs and skin fibroblasts derived from PBD-ZSD patients and healthy controls. Dermal fibroblast cultures from 2 orangutans were reprogrammed into iPSCs by transfection with retroviruses designed to express the human OCT4, SOX2, KLF4 and c-MYC cDNA. Fibroblasts and iPSCs were cultured in 1:1 ratio of DMEM:F12 medium supplemented with 20% KOSR (knock-out serum replacement) at 37°C with 5% CO2 until confluence for RNA extraction. The overall goal was to evaluate gene expression biomarkers of pluripotency in iPSCs and original fibroblast cultures.
Project description:Human naïve ESCs cultured in RSET medium with KLF17 knockdown and human primed ESCs cultured in mTeSR with KLF17 overexpression were sequenced by poly(A)+RNA-seq.