Ex vivo reconstitution of arterial endothelium by embryonic stem cell-derived endothelial progenitor cells in baboons.

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts downregulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.

Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm.

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a man's life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Human amniotic epithelial cells are reprogrammed more efficiently by induced pluripotency than adult fibroblasts.

Cellular reprogramming from adult somatic cells into an embryonic cell-like state, termed induced pluripotency, has been achieved in several cell types. However, the ability to reprogram human amniotic epithelial cells (hAECs), an abundant cell source derived from discarded placental tissue, has only recently been investigated. Here we show that not only are hAECs easily reprogrammed into induced pluripotent stem cells (AE-iPSCs), but hAECs reprogram faster and more efficiently than adult and neonatal somatic dermal fibroblasts. Furthermore, AE-iPSCs express higher levels of NANOG and OCT4 compared to human foreskin fibroblast iPSCs (HFF1-iPSCs) and express decreased levels of genes associated with differentiation, including NEUROD1 and SOX17, markers of neuronal differentiation. To elucidate the mechanism behind the higher reprogramming efficiency of hAECs, we analyzed global DNA methylation, global histone acetylation, and the mitochondrial DNA A3243G point mutation. Whereas hAECs show no differences in global histone acetylation or mitochondrial point mutation accumulation compared to adult and neonatal dermal fibroblasts, hAECs demonstrate a decreased global DNA methylation compared to dermal fibroblasts. Likewise, quantitative gene expression analyses show that hAECs endogenously express OCT4, SOX2, KLF4, and c-MYC, all four factors used in cellular reprogramming. Thus, hAECs represent an ideal cell type for testing novel approaches for generating clinically viable iPSCs and offer significant advantages over postnatal cells that more likely may be contaminated by environmental exposures and infectious agents.

Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages. METHODS: Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1ß, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines. RESULTS: HIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1ß, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1ß and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1ß and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1ß, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1ß and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors. CONCLUSION: Collectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.

mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells, including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate, as well as reversing those decisions during induced pluripotency (iPS), have focused largely on transcriptomic controls. Here, we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore, p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However, expression of constitutively active p70 S6K, but not wild-type p70 S6K, induces differentiation. Additionally, hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation.

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.

Characterization, cryopreservation, and ablation of spermatogonial stem cells in adult rhesus macaques.

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells.

As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).

Magnetic resonance imaging of embryonic and fetal development in model systems.

We give an overview of the applications and methods of high-resolution anatomical magnetic resonance imaging (MRI) in the study of embryonic and fetal development in animal models. Challenges associated with performing in utero studies are described. Recent in utero images in mouse and in nonhuman primates are presented. Results using magnetic resonance microscopy in fixed mouse embryos and in amphibian embryos in vivo are reviewed. We discuss how studies of pregnancy in animal models aid in the translation of innovative new MRI techniques to clinical applications.

Lamin A/C expression is a marker of mouse and human embryonic stem cell differentiation.

Nuclear lamins comprise the nuclear lamina, a scaffold-like structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription-polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.

The expression of mitochondrial DNA transcription factors during early cardiomyocyte in vitro differentiation from human embryonic stem cells.

Mitochondrial biogenesis and activation of both oxidative phosphorylation, as well as transcription and replication of the mitochondrial genome, are key regulatory events in cell differentiation. Mitochondrial DNA transcription and replication are highly dependent on the interaction with nuclear-encoded transcription factors translocated from the nucleus. Using a human embryonic stem cell line, HSF 6, we analyzed the proliferation of mitochondria and the expression of mtDNA-specific transcription factors in undifferentiated, migratory embryonic stem cells and spontaneously derived cardiomyocytes. Mitochondrial proliferation and mtDNA transcription are initiated in human embryonic stem cells as they undergo spontaneous differentiation in culture into beating cardiomyocytes. Undifferentiated, pluripotent human embryonic stem cells have few mitochondria, and, as they differentiate, they polarize to one extremity of the cell and then bipolarize the differentiating cell. The differentiated cell then adopts the cytoplasmic configuration of a somatic cell as evidenced in differentiating cardiomyocytes. Transcription and replication of the extranuclear mitochondrial genome is dependent on nuclear encoded factors exported to the mitochondrion. However, the differentiating cardiomyocytes have reduced or absent levels of these transcription and replication factors, namely mitochondrial transcription factors A, B1, B2, and nuclear respiratory factor 1 and polymerase gamma. Therefore, final embryonic stem cell commitment may be influenced by mitochondrial proliferation and mtDNA transcription. However, it is likely that differentiating cardiomyocytes are in mitochondrial arrest, awaiting commitment to a final cell fate.

Genome-scale expression profiling of Hutchinson-Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease with widespread phenotypic features resembling premature aging. HGPS was recently shown to be caused by dominant mutations in the LMNA gene, resulting in the in-frame deletion of 50 amino acids near the carboxyl terminus of the encoded lamin A protein. Children with this disease typically succumb to myocardial infarction or stroke caused by severe atherosclerosis at an average age of 13 years. To elucidate further the molecular pathogenesis of this disease, we compared the gene expression patterns of three HGPS fibroblast cell strains heterozygous for the LMNA mutation with three normal, age-matched cell strains. We defined a set of 361 genes (1.1% of the approximately 33,000 genes analysed) that showed at least a 2-fold, statistically significant change. The most prominent categories encode transcription factors and extracellular matrix proteins, many of which are known to function in the tissues severely affected in HGPS. The most affected gene, MEOX2/GAX, is a homeobox transcription factor implicated as a negative regulator of mesodermal tissue proliferation. Thus, at the gene expression level, HGPS shows the hallmarks of a developmental disorder affecting mesodermal and mesenchymal cell lineages. The identification of a large number of genes implicated in atherosclerosis is especially valuable, because it provides clues to pathological processes that can now be investigated in HGPS patients or animal models.

Safeguarding ART.

Assisted reproductive technologies (ART) are exceptional among clinical therapies, as unlike most medical procedures, ART have generational consequences. Further, human embryo research in the US has been sponsored solely by the private sector and, until recent biotechnology forays into human embryonic stem cell (hESC) and cloning research, exclusively by infertility clinics. Additionally, the relatively brief clinical history of ART has made it difficult for practitioners and researchers to agree on criteria for its safety and success. Against this backdrop, market pressure on biotechnology companies to create hESC lines and on clinical practices to occupy the innovative forefront has resulted in arguably risky experiments with human embryo cloning, as well as in unintentional germ-line genetic modifications during ART and perhaps during gene therapy. Reproduction, once governed largely by passions and instinct, now seems to need further governance. Some argue that it could now be time for the biomedical community, especially in the US, to take further steps to safeguard ART.