Parsing the pluripotency continuum in humans and non-human primates for interspecies chimera generation.

Pluripotency refers to the potential of single cells to form all cells and tissues of an organism. The observation that pluripotent stem cells can chimerize the embryos of evolutionarily distant species, albeit at very low efficiencies, could with further modifications, facilitate the production of human-animal interspecies chimeras. The generation of human-animal interspecies chimeras, if achieved, will enable practitioners to recapitulate pathologic human tissue formation in vivo and produce patient-specific organs inside livestock species. However, little is known about the nature of chimera-competent cellular states in primates. Here, I discuss recent advances in our understanding of the pluripotency continuum in humans and non-human primates (NHPs). Although undefined differences between humans and NHPs still justify the utility of studying human cells, the complementary use of NHP PS cells could also allow one to conduct pilot studies testing interspecies chimera generation strategies with reduced ethical concerns associated with human interspecies neurological chimerism. However, the availability of standardized, high-quality and validated NHP PS cell lines covering the spectrum of primate pluripotent states is lacking. Therefore, a clearer understanding of the primate pluripotency continuum will facilitate the complementary use of both human and NHP PS cells for testing interspecies organogenesis strategies, with the hope of one day enabling human organ generation inside livestock species.

Embryonic Chimeras with Human Pluripotent Stem Cells.

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the "holy grail" of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Neural Stem Cell Transplantation into a Mouse Model of Stroke.

Stroke is the fifth leading cause of death among Americans each year. Current standard-of-care treatment for stroke deploys intravenous tissue-type plasminogen activator (tPA), mechanical thrombolysis, or delivery of fibrinolytics. Although these therapies have reduced stroke-induced damage, therapeutic options still remain limited. Transplantation of patient-specific neural stem (NS) cells represents a promising strategy for the treatment of stroke. Basic science research has shown that transplanted NS cells can differentiate in the brain of rodent models of stroke and promote behavioral recovery. Clinical trials exploring the feasibility of stem cell treatment for stroke are currently being conducted. However, questions remain regarding the optimal means of delivering NS cells, including cell dose, infusion speed, timing of transplantation, anatomic site, and imaging-assisted monitoring and guidance. Of the different available delivery modalities, intravascular NS delivery after stroke represents one practical approach. In this chapter, I provide methods for intravascular delivery of NS cells in a mouse model of stroke. The techniques involved include cell culture of NS cells, flow cytometry of NS cells, modeling stroke via unilateral common carotid artery occlusion, intra-arterial injection of NS cells into the brain, behavior analyses, and immunohistochemistry. Intra-arterial NS cell therapy has the potential to improve functional recovery after ischemic stroke.

Human-Monkey Chimeras for Modeling Human Disease: Opportunities and Challenges.

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem cells (PS cells) and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. Here, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could surpass the limitations of current monkey models of neurological and psychiatric disease, but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Human-Monkey Chimeras for Modeling Human Disease: Opportunities and Challenges.

The search for a better animal model to simulate human disease has been a "holy grail" of biomedical research for decades. Recent identification of different types of pluripotent stem (PS) cells and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. In this study, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could not only surpass the limitations of current monkey models of neurological and psychiatric disease but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Generating Human Organs via Interspecies Chimera Formation: Advances and Barriers.

The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.

Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Distinct and combinatorial functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in mouse embryonic stem cell identity.

Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.

Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition.

During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.

Genome-wide profiling of pluripotent cells reveals a unique molecular signature of human embryonic germ cells.

Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency.

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA.

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells.

Accessing naïve human pluripotency.

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the naïve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether naïve human pluripotent stem cells with bona fide features of both a pluripotent state and naïve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of naïve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture naïve pluripotency in human cells. Unraveling how to stably induce naïve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.

Intravascular cell replacement therapy for stroke.

The use of stem cell transplantation to restore neurological function after stroke is being recognized as a potential novel therapy. Before stem cell transplantation can become widely applicable, however, questions remain about the optimal site of delivery and timing of transplantation. In particular, there seems to be increasing evidence that intravascular cell delivery after stroke is a viable alternative to intracerebral transplantation. In this review, the authors focus on the intravascular delivery of stem cells for stroke treatment with an emphasis on timing, transendothelial migration and possible mechanisms leading to neuroprotection, angiogenesis, immunomodulation, and neural plasticity. They also review current concepts of in vivo imaging and tracking of stem cells after stroke.

Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.

BACKGROUND AND PURPOSE: Intravascular delivery of neural stem cells (NSCs) after stroke has been limited by the low efficiency of transendothelial migration. Vascular cell adhesion molecule-1 is an endothelial adhesion molecule known to be upregulated early after stroke and is responsible for the firm adhesion of inflammatory cells expressing the surface integrin, CD49d. We hypothesize that enriching for NSCs that express CD49d and injecting them into the carotid artery would improve targeted cell delivery to the injured brain. METHODS: Mouse NSCs were analyzed for the expression of CD49d by fluorescence activated cell sorting. A CD49d-enriched (CD49d(+)) (>95%) and -depleted (CD49d(-); <5%) NSC population was obtained by cell sorting. C57/Bl6 mice underwent left-sided hypoxia-ischemia surgery and were assigned to receive 3 x 10(5) CD49d(+), CD49d(-) NSCs, or vehicle injection into the left common carotid artery 48 hours after stroke. Behavioral recovery was measured using a rotarod for 2 weeks after cell injection. RESULTS: Fluorescence activated cell sorting analysis revealed 25% CD49d(+) NSCs. In a static adhesion assay, NSCs adhered to vascular cell adhesion molecule-1 in a dose-dependent manner. Significantly more NSCs were found in the cortex, the hippocampus, and the subventricular zone in the ischemic hemisphere in animals receiving CD49d(+) NSCs as compared with CD49d(-) NSCs (P<0.05). Animals treated with CD49d(+) cells showed a significantly better behavioral recovery as compared with CD49d(-) and vehicle-treated animals. CONCLUSIONS: We show that enrichment of NSCs by fluorescence activated cell sorting for the surface integrin, CD49d, and intracarotid delivery promotes cell homing to the area of stroke in mice and improves behavioral recovery.

Neural progenitor cells transplanted into the uninjured brain undergo targeted migration after stroke onset.

Endogenous neural stem cells normally reside in their niche, the subventricular zone, in the uninjured rodent brain. Upon stroke, these cells become more proliferative and migrate away from the subventricular zone into the surrounding parenchyma. It is not known whether this stroke-induced behavior is due to changes in the niche or introduction of attractive cues in the infarct zone, or both. A related question is how transplanted neural stem cells respond to subsequent insults, including whether exogenous stem cells have the plasticity to respond to subsequent injuries after engraftment. We addressed this issue by transplanting neural progenitor cells (NPCs) into the uninjured brain and then subjecting the animal to stroke. We were able to follow the transplanted NPCs in vivo by labeling them with superparamagnetic iron oxide particles and imaging them via high-resolution magnetic resonance imaging (MRI) during engraftment and subsequent to stroke. We find that transplanted NPCs that are latent can be activated in response to stroke and exhibit directional migration into the parenchyma, similar to endogenous neural NPCs, without a niche environment.