Using embryo models to understand the development and progression of embryonic lineages: a focus on primordial germ cell development.

BACKGROUND: Recapitulating mammalian cell type differentiation in vitro promises to improve our understanding of how these processes happen in vivo, while bringing additional prospects for biomedical applications. The establishment of stem cell-derived embryo models and embryonic organoids, which have experienced explosive growth over the last few years, open new avenues for research due to their scale, reproducibility, and accessibility. Embryo models mimic various developmental stages, exhibit different degrees of complexity, and can be established across species. Since embryo models exhibit multiple lineages organised spatially and temporally, they are likely to provide cellular niches that, to some degree, recapitulate the embryonic setting and enable "co-development" between cell types and neighbouring populations. One example where this is already apparent is in the case of primordial germ cell-like cells (PGCLCs). SUMMARY: While directed differentiation protocols enable the efficient generation of high PGCLC numbers, embryo models provide an attractive alternative as they enable the study of interactions of PGCLCs with neighbouring cells, alongside the regulatory molecular and biophysical mechanisms of PGC competency. Additionally, some embryo models can recapitulate post-specification stages of PGC development (including migration or gametogenesis), mimicking the inductive signals pushing PGCLCs to mature and differentiate, and enabling the study of PGCLC development across stages. Therefore, in vitro models may allow us to address questions of cell type differentiation, and PGC development specifically, that have hitherto been out of reach with existing systems. KEY MESSAGE: This review evaluates the current advances in stem cell-based embryo models, with a focus on their potential to model cell type-specific differentiation in general, and in particular to address open questions in PGC development and gametogenesis.

Transgene-Free Cynomolgus Monkey iPSCs Generated under Chemically Defined Conditions.

Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.

Generation of marmoset primordial germ cell-like cells under chemically defined conditions.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and oocytes, which transmit genetic/epigenetic information across generations. Mouse PGC and subsequent gamete development can be fully reconstituted in vitro, opening up new avenues for germ cell studies in biomedical research. However, PGCs show molecular differences between rodents and humans. Therefore, to establish an in vitro system that is closely related to humans, we studied PGC development in vivo and in vitro in the common marmoset monkey Callithrix jacchus (cj). Gonadal cjPGCs at embryonic day 74 express SOX17, AP2Ɣ, BLIMP1, NANOG, and OCT4A, which is reminiscent of human PGCs. We established transgene-free induced pluripotent stem cell (cjiPSC) lines from foetal and postnatal fibroblasts. These cjiPSCs, cultured in defined and feeder-free conditions, can be differentiated into precursors of mesendoderm and subsequently into cjPGC-like cells (cjPGCLCs) with a transcriptome similar to human PGCs/PGCLCs. Our results not only pave the way for studying PGC development in a non-human primate in vitro under experimentally controlled conditions, but also provide the opportunity to derive functional marmoset gametes in future studies.

The cytokine receptor CRLF3 is a human neuroprotective EV-3 (Epo) receptor.

The evolutionary conserved orphan cytokine receptor-like factor 3 (CRLF3) has been implicated in human disease, vertebrate hematopoiesis and insect neuroprotection. While its specific functions are elusive, experimental evidence points toward a general role in cell homeostasis. Erythropoietin (Epo) is a major regulator of vertebrate hematopoiesis and a general cytoprotective cytokine. Erythropoietic functions mediated by classical Epo receptor are understood in great detail whereas Epo-mediated cytoprotective mechanisms are more complex due to involvement of additional Epo receptors and a non-erythropoietic splice variant with selectivity for certain receptors. In the present study, we show that the human CRLF3 mediates neuroprotection upon activation with the natural Epo splice variant EV-3. We generated CRLF3 knock-out iPSC lines and differentiated them toward the neuronal lineage. While apoptotic death of rotenone-challenged wild type iPSC-derived neurons was prevented by EV-3, EV-3-mediated neuroprotection was absent in CRLF3 knock-out neurons. Rotenone-induced apoptosis and EV-3-mediated neuroprotection were associated with differential expression of pro-and anti-apoptotic genes. Our data characterize human CRLF3 as a receptor involved in Epo-mediated neuroprotection and identify CRLF3 as the first known receptor for EV-3.

Exploring the Potential of Symmetric Exon Deletion to Treat Non-Ischemic Dilated Cardiomyopathy by Removing Frameshift Mutations in TTN.

Non-ischemic dilated cardiomyopathy (DCM) is one of the most frequent pathologies requiring cardiac transplants. Even though the etiology of this disease is complex, frameshift mutations in the giant sarcomeric protein Titin could explain up to 25% of the familial and 18% of the sporadic cases of DCM. Many studies have shown the potential of genome editing using CRISPR/Cas9 to correct truncating mutations in sarcomeric proteins and have established the grounds for myoediting. However, these therapies are still in an immature state, with only few studies showing an efficient treatment of cardiac diseases. This publication hypothesizes that the Titin (TTN)-specific gene structure allows the application of myoediting approaches in a broad range of locations to reframe TTNtvvariants and to treat DCM patients. Additionally, to pave the way for the generation of efficient myoediting approaches for DCM, we screened and selected promising target locations in TTN. We conceptually explored the deletion of symmetric exons as a therapeutic approach to restore TTN's reading frame in cases of frameshift mutations. We identified a set of 94 potential candidate exons of TTN that we consider particularly suitable for this therapeutic deletion. With this study, we aim to contribute to the development of new therapies to efficiently treat titinopathies and other diseases caused by mutations in genes encoding proteins with modular structures, e.g., Obscurin.

Non-human primate pluripotent stem cells for the preclinical testing of regenerative therapies.

Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies. Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases. Therefore, the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years. For the preclinical validation of cell replacement therapies in non-human primates, it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts. However, pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems. In recent years, however, relevant progress has also been made with non-human primate pluripotent stem cells. This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies. We focus on the critical domains of (1) reprogramming and embryonic stem cell line derivation, (2) cell line maintenance and characterization and, (3) application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.

Primate Simplexviruses Differ in Tropism for Macaque Cells.

Primate simplexviruses are closely related neurotropic herpesviruses, which are largely apathogenic in their respective host species. However, cross-species transmission of Macacine alphaherpesvirus 1 (McHV1, also termed herpes B virus) from rhesus macaques to humans can cause fatal encephalomyelitis. In contrast, closely related viruses, such as Cercopithecine alphaherpesvirus 2 (CeHV2, also termed simian agent 8) or Papiine alphaherpesvirus 2 (PaHV2, also termed herpesvirus papio 2), have not been linked to human disease and are believed to be largely apathogenic in humans. Here, we investigated whether McHV1, PaHV2 and CeHV2 differ in their capacity to infect human and non-human primate (NHP) cells. For comparison, we included the human simplexviruses HSV1 and HSV2 in our analyses. All five viruses replicated efficiently in cell lines of human and African green monkey origin, and McHV1 and PaHV2 also showed robust replication in rhesus macaque cell lines. In contrast, the replication of CeHV2 and particularly HSV1 and HSV2 in cell lines of rhesus macaque origin were reduced or inefficient. Similarly, McHV1, but not CeHV2, efficiently infected rhesus macaque brain organoids. These results point towards the previously unappreciated partial resistance of certain rhesus macaque cells to HSV1/HSV2/CeHV2 infection and reveal similarities between the cell tropism of McHV1 and PaHV2 that might be relevant for risk assessment.

Generation and Cultivation of Transgene-Free Macaque and Baboon iPSCs Under Chemically Defined Conditions.

Non-human primates (NHP), and in particular Old World monkeys including macaques and baboons, are key animal models for the late preclinical testing of novel stem cell-based therapies and other advanced therapy medical products (ATMP) for the treatment of degenerative diseases. These pathologies are characterized by the loss of functional cells in an organ, as in Parkinson's disease, age-related macular degeneration, or after myocardial infarction. For preclinically relevant testing of induced pluripotent stem cell (iPSC)-based therapies, robust, and standardized protocols for the generation, characterization, and differentiation of NHP-iPSCs are required. Since the discovery of iPSCs by Takahashi and Yamanaka in 2006, human reprogramming protocols have been continuously refined. However, the generation of integration-free NHP-iPSC lines and a stable feeder- and serum-free long-term culture turned out to be difficult or even impossible with the current protocols established for human iPSCs. Here, we provide a robust protocol for the generation of transgene-free Old World monkey (and human) iPSCs and long-term cultivation under chemically defined conditions. This protocol was successfully applied to generate human, baboon (Papio anubis), rhesus (Macaca mulatta), and cynomolgus macaque (Macaca fascicularis) iPSCs from skin fibroblasts. The resulting NHP-iPSCs provide a valuable resource for the preclinical testing of regenerative therapies in NHP.

A piggyBac-based platform for genome editing and clonal rhesus macaque iPSC line derivation.

Non-human primates (NHPs) are, due to their close phylogenetic relationship to humans, excellent animal models to study clinically relevant mutations. However, the toolbox for the genetic modification of NHPs is less developed than those for other species like mice. Therefore, it is necessary to further develop and refine genome editing approaches in NHPs. NHP pluripotent stem cells (PSCs) share key molecular signatures with the early embryo, which is an important target for genomic modification. Therefore, PSCs are a valuable test system for the validation of embryonic genome editing approaches. In the present study, we made use of the versatility of the piggyBac transposon system for different purposes in the context of NHP stem cell technology and genome editing. These include (1) Robust reprogramming of rhesus macaque fibroblasts to induced pluripotent stem cells (iPSCs); (2) Culture of the iPSCs under feeder-free conditions even after removal of the transgene resulting in transgene-free iPSCs; (3) Development of a CRISPR/Cas-based work-flow to edit the genome of rhesus macaque PSCs with high efficiency; (4) Establishment of a novel protocol for the derivation of gene-edited monoclonal NHP-iPSC lines. These findings facilitate efficient testing of genome editing approaches in NHP-PSC before their in vivo application.

SIRT1 Expression and Regulation in the Primate Testis.

The epigenetic mechanisms controlling germ cell development and differentiation are still not well understood. Sirtuin-1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase and belongs to the sirtuin family of deacetylases. It catalyzes the removal of acetyl groups from a number of protein substrates. Some studies reported a role of SIRT1 in the central and peripheral regulation of reproduction in various non-primate species. However, testicular SIRT1 expression and its possible role in the testis have not been analyzed in primates. Here, we document expression of SIRT1 in testes of different primates and some non-primate species. SIRT1 is expressed mainly in the cells of seminiferous tubules, particularly in germ cells. The majority of SIRT1-positive germ cells were in the meiotic and postmeiotic phase of differentiation. However, SIRT1 expression was also observed in selected premeiotic germ cells, i.e., spermatogonia. SIRT1 co-localized in spermatogonia with irisin, an endocrine factor specifically expressed in primate spermatogonia. In marmoset testicular explant cultures, SIRT1 transcript levels are upregulated by the addition of irisin as compared to untreated controls explants. Rhesus macaques are seasonal breeders with high testicular activity in winter and low testicular activity in summer. Of note, SIRT1 mRNA and SIRT1 protein expression are changed between nonbreeding (low spermatogenesis) and breeding (high spermatogenesis) season. Our data suggest that SIRT1 is a relevant factor for the regulation of spermatogenesis in primates. Further mechanistic studies are required to better understand the role of SIRT1 during spermatogenesis.

Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules.

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of cell-based therapies; however, the safety and efficacy of potential iPSC-based treatments need to be verified in relevant animal disease models before their application in the clinic. Here, we report the derivation of iPSCs from common marmoset monkeys (Callithrix jacchus) using self-replicating mRNA vectors based on the Venezuelan equine encephalitis virus (VEE-mRNAs). By transfection of marmoset fibroblasts with VEE-mRNAs carrying the human OCT4, KLF4, SOX2, and c-MYC and culture in the presence of small molecule inhibitors CHIR99021 and SB431542, we first established intermediate primary colonies with neural progenitor-like properties. In the second reprogramming step, we converted these colonies into transgene-free pluripotent stem cells by further culturing them with customized marmoset iPSC medium in feeder-free conditions. Our experiments revealed a novel paradigm for flexible reprogramming of somatic cells, where primary colonies obtained by a single VEE-mRNA transfection can be directed either toward the neural lineage or further reprogrammed to pluripotency. These results (1) will further enhance the role of the common marmoset as animal disease model for preclinical testing of iPSC-based therapies and (2) establish an in vitro system to experimentally address developmental signal transduction pathways in primates.

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

Baboon induced pluripotent stem cell generation by piggyBac transposition of reprogramming factors.

Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.

Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.

Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of "highly expressed equals high repression".

The ubiquitin ligase c-CBL is expressed in undifferentiated marmoset monkey pluripotent stem cells but is not a general stem cell marker.

The protein c-CBL is a ubiquitin ligase. It catalyzes the last step of the transfer of ubiquitin to target proteins. Upon completion of polyubiquitination, the target proteins are degraded. Clinically, it is important that c-CBL is mutated in a subset of patients who develop myeloid malignancies, which are diseases of the hematopoietic stem or progenitor cells. c-CBL has also been shown to be expressed by human spermatogonia. The whole spermatogonial cell population possesses a subset that comprises also the spermatogonial stem cells. Based on these findings we hypothesized that c-CBL might be a general stem cell marker. To test this, we first validated the antibody using marmoset bone marrow and adult testis. In both tissues, the expected staining pattern was observed. Western blot analysis revealed only one band of the expected size. Then, we examined the expression of c-CBL in marmoset monkey embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and adult stem cells. We found that c-CBL is strongly expressed in undifferentiated marmoset iPS cells and ES cells. However, adult stem cells in the gut and the stomach did not express c-CBL, indicating that c-CBL is not a general stem cell marker. In summary, c-CBL is strongly expressed in pluripotent stem cells of the marmoset monkey as well as in selected adult stem cell types. Future studies will define the function of c-CBL in pluripotent stem cells.