A Standardized Nomenclature Design for Systematic Referencing and Identification of Animal Cellular Material.

The documentation, preservation and rescue of biological diversity increasingly uses living biological samples. Persistent associations between species, biosamples, such as tissues and cell lines, and the accompanying data are indispensable for using, exchanging and benefiting from these valuable materials. Explicit authentication of such biosamples by assigning unique and robust identifiers is therefore required to allow for unambiguous referencing, avoid identification conflicts and maintain reproducibility in research. A predefined nomenclature based on uniform rules would facilitate this process. However, such a nomenclature is currently lacking for animal biological material. We here present a first, standardized, human-readable nomenclature design, which is sufficient to generate unique and stable identifying names for animal cellular material with a focus on wildlife species. A species-specific human- and machine-readable syntax is included in the proposed standard naming scheme, allowing for the traceability of donated material and cultured cells, as well as data FAIRification. Only when it is consistently applied in the public domain, as publications and inter-institutional samples and data are exchanged, distributed and stored centrally, can the risks of misidentification and loss of traceability be mitigated. This innovative globally applicable identification system provides a standard for a sustainable structure for the long-term storage of animal bio-samples in cryobanks and hence facilitates current as well as future species conservation and biomedical research.

Dual effects of ARX poly-alanine mutations in human cortical and interneuron development.

Mutations in ARX , an X-linked gene, are implicated in a wide spectrum of neurological disorders including patients who have intellectual disability and epilepsy. Mouse models have shown that Arx is critical for cortical development and interneuron migration, however they do not recapitulate the full phenotype observed in patients. Moreover, the epilepsy in many patients with poly-alanine tract expansion (PAE) mutations in ARX show pharmacoresistance, emphasizing the need to develop new treatments. Here, we used human neural organoid models to study the consequences of PAE mutations, one of the most prevalent mutations in ARX . We found that PAE mutations result in an early increase in radial glia cells and intermediate progenitor cells, and premature differentiation leading to a loss of cortical neurons at later timepoints. Moreover, ARX expression is upregulated in CO derived from patient at 30 DIV which alters the expression of CDKN1C , SFRP1 , DLK1 and FABP7 , among others. We also found a cell autonomously enhanced interneuron migration, which can be rescued by CXCR4 inhibition. Furthermore, ARX PAE assembloids had hyper-activity and synchrony evident from the early stages. These data provide novel insights to the pathogenesis of these and likely related human neurological disorders and identifies a critical window for therapeutic interventions.

Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells.

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.

Validation of baboon pluripotent cells as a model for translational stem cell research.

Translation of stem cell therapies to the clinic will be most successful following optimization of efficacy and safety in appropriate preclinical model systems. Among available models, nonhuman primates (NHPs) provide the most accurate recapitulation of human anatomy, physiology, genetics and epigenetics. Here, we show that baboon pluripotent cells (PSCs) recapitulate key molecular features of human PSCs with greater accuracy than that found in PSCs from non-primate species such as mice. Specifically, baboon and human PSCs exhibit greater conservation of gene expression patterns, higher sequence and structural homology among pluripotency factors, more equivalent genome-wide patterns of histone and DNA methylation modifications, and similar maintenance of bivalent programming of developmental genes than that found between human and non-primate PSCs.

Regeneration enhanced in critical-sized bone defects using bone-specific extracellular matrix protein.

Extracellular matrix (ECM) products have the potential to improve cellular attachment and promote tissue-specific development by mimicking the native cellular niche. In this study, the therapeutic efficacy of an ECM substratum produced by bone marrow stem cells (BM-MSCs) to promote bone regeneration in vitro and in vivo were evaluated. Fluorescence-activated cell sorting analysis and phenotypic expression were employed to characterize the in vitro BM-MSC response to bone marrow specific ECM (BM-ECM). BM-ECM encouraged cell proliferation and stemness maintenance. The efficacy of BM-ECM as an adjuvant in promoting bone regeneration was evaluated in an orthotopic, segmental critical-sized bone defect in the rat femur over 8 weeks. The groups evaluated were either untreated (negative control); packed with calcium phosphate granules or granules+BM-ECM free protein and stabilized by collagenous membrane. Bone regeneration in vivo was analyzed using microcomputed tomography and histology. in vivo results demonstrated improvements in mineralization, osteogenesis, and tissue infiltration (114 ± 15% increase) in the BM-ECM complex group from 4 to 8 weeks compared to mineral granules only (45 ± 21% increase). Histological observations suggested direct apposition of early bone after 4 weeks and mineral consolidation after 8 weeks implantation for the group supplemented with BM-ECM. Significant osteoid formation and greater functional bone formation (polar moment of inertia was 71 ± 0.2 mm4 with BM-ECM supplementation compared to 48 ± 0.2 mm4 in untreated defects) validated in vivo indicated support of osteoconductivity and increased defect site cellularity. In conclusion, these results suggest that BM-ECM free protein is potentially a therapeutic supplement for stemness maintenance and sustaining osteogenesis.

Fertilization and Cleavage Axes Differ In Primates Conceived By Conventional (IVF) Versus Intracytoplasmic Sperm Injection (ICSI).

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

Optimization of culture conditions for the derivation and propagation of baboon (Papio anubis) induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) offer the possibility of cell replacement therapies using patient-matched cells to treat otherwise intractable diseases and debilitations. To successfully realize this potential, several factors must be optimized including i) selection of the appropriate cell type and numbers to transplant, ii) determination of the means of transplantation and the location into which the transplanted cells should be delivered, and iii) demonstration of the safety and efficacy of the cell replacement protocol to mitigate each targeted disease state. A majority of diseases or debilitations likely to be targeted by cell-based therapeutic approaches represent complex conditions or physiologies manifest predominantly in primates including humans. Nonhuman primates afford the most clinically relevant model system for biomedical studies and testing of cell-based therapies. Baboons have 92% genomic similarity with humans overall and especially significant similarities in their immunogenetic system, rendering this species a particularly valuable model for testing procedures involving cell transplants into living individuals. To maximize the utility of the baboon model, standardized protocols must be developed for the derivation of induced pluripotent stem cells from living adults and the long-term maintenance of these cells in culture. Here we tested four commercially available culture systems (ReproFF, mTeSR1, E8 and Pluristem) for competence to maintain baboon iPSCs in a pluripotent state over multiple passages, and to support the derivation of new lines of baboon iPSCs. Of these four media only Pluristem was able to maintain baboon pluripotency as assessed by morphological characteristics, immunocytochemistry and RT-qPCR. Pluristem also facilitated the derivation of new lines of iPSCs from adult baboon somatic cells, which had previously not been accomplished. We derived multiple iPS cell lines from adult baboon peripheral blood mononuclear cells cultured in Pluristem. These were validated by expression of the pluripotency markers OCT4, NANOG, SOX2, SSEA4 and TRA181, as well as the ability to differentiate into tissues from all three germ layers when injected into immunocompromised mice. These findings further advance the utility of the baboon as an ideal preclinical model system for optimizing iPS cell-based, patient-specific replacement therapies in humans.

Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killing.

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guerin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24-48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.

Advantages of nonhuman primates as preclinical models for evaluating stem cell-based therapies for Parkinson's disease.

The derivation of dopaminergic neurons from induced pluripotent stem cells brings new hope for a patient-specific, stem cell-based replacement therapy to treat Parkinson's disease (PD) and related neurodegenerative diseases; and this novel cell-based approach has already proven effective in animal models. However, there are several aspects of this procedure that have yet to be optimized to the extent required for translation to an optimal cell-based transplantation protocol in humans. These challenges include pinpointing the optimal graft location, appropriately scaling up the graft volume, and minimizing the risk of chronic immune rejection, among others. To advance this procedure to the clinic, it is imperative that a model that accurately and fully recapitulates characteristics most pertinent to a cell-based transplantation to the human brain is used to optimize key technical aspects of the procedure. Nonhuman primates mimic humans in multiple ways including similarities in genomics, neuroanatomy, neurophysiology, immunogenetics, and age-related changes in immune function. These characteristics are critical to the establishment of a relevant model in which to conduct preclinical studies to optimize the efficacy and safety of cell-based therapeutic approaches to the treatment of PD. Here we review previous studies in rodent models, and emphasize additional advantages afforded by nonhuman primate models in general, and the baboon model in particular, for preclinical optimization of cell-based therapeutic approaches to the treatment of PD and other neurodegenerative diseases. We outline current unresolved challenges to the successful application of stem cell therapies in humans and propose that the baboon model in particular affords a number of traits that render it most useful for preclinical studies designed to overcome these challenges.

Contribution of FcɛRI-associated vesicles to mast cell-macrophage communication following Francisella tularensis infection.

Understanding innate immune intercellular communication following microbial infection remains a key biological issue. Using live cell imaging, we demonstrate that mast cells actively extend cellular projections to sample the macrophage periphery during Francisella tularensis LVS infection. Mast cell MHCII(hi) expression was elevated from less than 1% to 13% during LVS infection. Direct contact during co-culture with macrophages further increased mast cell MHCII(hi) expression to approximately 87%. Confocal analyses of the cellular perimeter revealed mast cell caspase-1 was localized in close proximity with FcɛRI in uninfected mast cells, and repositioned to clustered regions upon LVS infection. Importantly, mast cell FcɛRI-encompassed vesicles are transferred to macrophages by trogocytosis, and macrophage caspase-1 expression is further up-regulated upon direct contact with mast cells. Our study reveals direct cellular interactions between innate cells that may impact the function of caspase-1, a known sensor of microbial danger and requirement for innate defense against many pathogenic microbes including F. tularensis.

Differentiation and Characterization of Dopaminergic Neurons From Baboon Induced Pluripotent Stem Cells.

UNLABELLED: : The progressive death of dopamine producing neurons in the substantia nigra pars compacta is the principal cause of symptoms of Parkinson's disease (PD). Stem cells have potential therapeutic use in replacing these cells and restoring function. To facilitate development of this approach, we sought to establish a preclinical model based on a large nonhuman primate for testing the efficacy and safety of stem cell-based transplantation. To this end, we differentiated baboon fibroblast-derived induced pluripotent stem cells (biPSCs) into dopaminergic neurons with the application of specific morphogens and growth factors. We confirmed that biPSC-derived dopaminergic neurons resemble those found in the human midbrain based on cell type-specific expression of dopamine markers TH and GIRK2. Using the reverse transcriptase quantitative polymerase chain reaction, we also showed that biPSC-derived dopaminergic neurons express PAX6, FOXA2, LMX1A, NURR1, and TH genes characteristic of this cell type in vivo. We used perforated patch-clamp electrophysiology to demonstrate that biPSC-derived dopaminergic neurons fired spontaneous rhythmic action potentials and high-frequency action potentials with spike frequency adaption upon injection of depolarizing current. Finally, we showed that biPSC-derived neurons released catecholamines in response to electrical stimulation. These results demonstrate the utility of the baboon model for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches for the treatment of PD. SIGNIFICANCE: Functional dopamine neurons were produced from baboon induced pluripotent stem cells, and their properties were compared to baboon midbrain cells in vivo. The baboon has advantages as a clinically relevant model in which to optimize the efficacy and safety of stem cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. Baboons possess crucial neuroanatomical and immunological similarities to humans, and baboon pluripotent stem cells can be differentiated into functional neurons that mimic those in the human brain, thus laying the foundation for the utility of the baboon model for evaluating stem cell therapies.

M-Cells Contribute to the Entry of an Oral Vaccine but Are Not Essential for the Subsequent Induction of Protective Immunity against Francisella tularensis.

M-cells (microfold cells) are thought to be a primary conduit of intestinal antigen trafficking. Using an established neutralizing anti-RANKL (Receptor Activator of NF-κB Ligand) antibody treatment to transiently deplete M-cells in vivo, we sought to determine whether intestinal M-cells were required for the effective induction of protective immunity following oral vaccination with ΔiglB (a defined live attenuated Francisella novicida mutant). M-cell depleted, ΔiglB-vaccinated mice exhibited increased (but not significant) morbidity and mortality following a subsequent homotypic or heterotypic pulmonary F. tularensis challenge. No significant differences in splenic IFN-γ, IL-2, or IL-17 or serum antibody (IgG1, IgG2a, IgA) production were observed compared to non-depleted, ΔiglB-vaccinated animals suggesting complementary mechanisms for ΔiglB entry. Thus, we examined other possible routes of gastrointestinal antigen sampling following oral vaccination and found that ΔiglB co-localized to villus goblet cells and enterocytes. These results provide insight into the role of M-cells and complementary pathways in intestinal antigen trafficking that may be involved in the generation of optimal immunity following oral vaccination.

Differential localization and high expression of SURVIVIN splice variants in human embryonic stem cells but not in differentiated cells implicate a role for SURVIVIN in pluripotency.

The BIRC5 gene encodes the oncofetal protein SURVIVIN, as well as four additional splice variants (ΔEx3, 2B, 3B and 2α). SURVIVIN, an inhibitor of apoptosis, is also a chromosomal passenger protein (CPP). Previous results have demonstrated that SURVIVIN is expressed at high levels in embryonic stem cells and inhibition of SURVIVIN function results in apoptosis, however these studies have not investigated the other four splice variants. In this study, we demonstrate that all variants are expressed at significantly higher levels in human embryonic stem (hES) cells than in differentiated cells. We examined the subcellular localization of the three most highly expressed variants. SURVIVIN displayed canonical CPP localization in mitotic cells and cytoplasmic localization in interphase cells. In contrast, SURVIVIN-ΔEx3 and SURVIVIN-2B did not localize as a CPP; SURVIVIN-ΔEx3 was found constitutively in the nucleus while SURVIVIN-2B was distributed along the chromosomes during mitosis and also to the mitotic spindle poles. We used inducible shRNA against SURVIVIN to inhibit expression in a titratable fashion. Using this system, we reduced the mRNA levels of these three variants to approx. 40%, resulting in a concomitant reduction of OCT4 and NANOG mRNA, suggesting a role for the SURVIVIN variants in pluripotency.

Derivation of induced pluripotent stem cells from the baboon: a nonhuman primate model for preclinical testing of stem cell therapies.

Development of effective pluripotent stem cell-based therapies will require safety and efficacy testing in a clinically relevant preclinical model such as nonhuman primates (NHPs). Baboons and macaques are equally similar to humans genetically and both have been extensively used for biomedical research. Macaques are preferred for human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) research whereas baboons are preferred for transplantation studies because of the greater similarity of their anatomy and immunogenetic system to those of humans. We generated four induced pluripotent stem cell (iPSC) lines from skin cells of the olive baboon (Papio anubis). Each line shows the distinct morphology of primate pluripotent stem cells, including flat colonies with well-defined borders and a high nuclear/cytoplasm ratio. Each is positive for the pluripotency markers OCT4, SOX2, NANOG, and SSEA4. Pluripotency was confirmed in two lines by teratoma formation with representative tissues from each germ layer, whereas a third produced cells from all three germ layers following embryoid body differentiation. Three lines have a normal male karyotype and the fourth is missing the short arm of one copy of chromosome 18. This may serve as an in vitro model for the human developmental disorder 18p-, which impacts 1 in 50,000 births/year. These iPSC lines represent the first step toward establishing the baboon as a NHP model for developing stem cell-based therapies.

Mesenchymal stem cell osteodifferentiation in response to alternating electric current.

The present study addressed adult human mesenchymal stem cell (MSC) differentiation toward the osteoblastic lineage in response to alternating electric current, a biophysical stimulus. For this purpose, MSCs (chosen because of their proven capability for osteodifferentiation in the presence of select bone morphogenetic proteins) were dispersed and cultured within electric-conducting type I collagen hydrogels, in the absence of supplemented exogenous dexamethasone and/or growth factors, and were exposed to either 10 or 40 µA alternating electric current for 6 h per day. Under these conditions, MSCs expressed both early- (such as Runx-2 and osterix) and late- (specifically, osteopontin and osteocalcin) osteogenic genes as a function of level, and duration of exposure to alternating electric current. Compared to results obtained after 7 days, gene expression of osteopontin and osteocalcin (late-osteogenic genes) increased at day 14. In contrast, expression of these osteogenic markers from MSCs cultured under similar conditions and time periods, but not exposed to alternating electric current, did not increase as a function of time. Most importantly, expression of genes pertinent to the either adipogenic (specifically, Fatty Acid Binding Protein-4) or chondrogenic (specifically, type II collagen) pathways was not detected when MSCs were exposed to the aforementioned alternating electric-current conditions tested in the present study. The present research study was the first to provide evidence that alternating electric current promoted the differentiation of adult human MSCs toward the osteogenic pathway. Such an approach has the yet untapped potential to provide critically needed differentiated cell supplies for cell-based assays and/or therapies for various biomedical applications.

Mast cell TLR2 signaling is crucial for effective killing of Francisella tularensis.

TLR signaling is critical for early host defense against pathogens, but the contributions of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection are largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the live vaccine strain were used to investigate the contribution of mast cell/TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHC class II and lysosomal-associated membrane protein 2. Infected TLR2(-/-) mast cells, in contrast to wild-type and TLR4(-/-) cells, lacked detectable IL-4 and displayed increased cell death with a 2-3 log increase of F. tularensis replication, but could be rescued with rIL-4 treatment. Importantly, MHC class II and lysosomal-associated membrane protein 2 localization with labeled F. tularensis in the lungs was greater in wild-type than in TLR2(-/-) mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.

Cell cycle adaptations and maintenance of genomic integrity in embryonic stem cells and induced pluripotent stem cells.

Pluripotent stem cells have the capability to undergo unlimited self-renewal and differentiation into all somatic cell types. They have acquired specific adjustments in the cell cycle structure that allow them to rapidly proliferate, including cell cycle independent expression of cell cycle regulators and lax G(1) to S phase transition. However, due to the developmental role of embryonic stem cells (ES) it is essential to maintain genomic integrity and prevent acquisition of mutations that would be transmitted to multiple cell lineages. Several modifications in DNA damage response of ES cells accommodate dynamic cycling and preservation of genetic information. The absence of a G(1)/S cell cycle arrest promotes apoptotic response of damaged cells before DNA changes can be fixed in the form of mutation during the S phase, while G(2)/M cell cycle arrest allows repair of damaged DNA following replication. Furthermore, ES cells express higher level of DNA repair proteins, and exhibit enhanced repair of multiple types of DNA damage. Similarly to ES cells, induced pluripotent stem (iPS) cells are poised to proliferate and exhibit lack of G(1)/S cell cycle arrest, extreme sensitivity to DNA damage, and high level of expression of DNA repair genes. The fundamental mechanisms by which the cell cycle regulates genomic integrity in ES cells and iPS cells are similar, though not identical.

Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts.

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.

Pluripotency genes overexpressed in primate embryonic stem cells are localized on homologues of human chromosomes 16, 17, 19, and X.

While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X, rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genes overexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiated progeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whom gametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentially on rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiated parental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are genetically identical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicated in neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derived from prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinically discarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions in pluripotency and suggests specific candidates for targeting differentiation and transformation decisions.

Evaluating protocols for embryonic stem cell differentiation into insulin-secreting beta-cells using insulin II-GFP as a specific and noninvasive reporter.

Stable and full differentiation of pluripotent stem cells into functional beta-cells offers the potential to treat type I diabetes with a theoretically inexhaustible source of replacement cells. In addition to the difficulties in directed differentiation, progress toward an optimized and reliable protocol has been hampered by the complication that cultured cells will concentrate insulin from the media, thus making it difficult to tell which, if any, cells are producing insulin. To address this, we utilized a novel murine embryonic stem cell (mESC) research model, in which the green fluorescent protein (GFP) has been inserted within the C-peptide of the mouse insulinII gene (InsulinII-GFP). Using this method, cells producing insulin are easily identified. We then compared four published protocols for differentiating mESCs into beta-cells to evaluate their relative efficiency by assaying intrinsic insulin production. Cells differentiated using each protocol were easily distinguished based on culture conditions and morphology. This comparison is strengthened because all testing is performed within the same laboratory by the same researchers, thereby removing interlaboratory variability in culture, cells, or analysis. Differentiated cells were analyzed and sorted based on GFP fluorescence as compared to wild type cells. Each differentiation protocol increased GFP fluorescence but only modestly. None of these protocols yielded more than 3% of cells capable of insulin biosynthesis indicating the relative inefficiency of all analyzed protocols. Therefore, improved beta-cells differentiation protocols are needed, and these insulin II GFP cells may prove to be an important tool to accelerate this process.