Generation of germline-competent rat induced pluripotent stem cells

Background

Recent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.

Methodology/Principal Findings

We generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.

Conclusions/Significance

Our data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.     

Establishment of a novel method for the production of chimeric mouse embryos using water-in-oil droplets

Production of chimeric animals is often a necessity for the generation of genetically modified animals and has gained popularity in recent years in regenerative medicine for the reconstruction of xenogeneic organs. Aggregation and injection methods are generally used to produce chimeric mice. In the aggregation method, the chimeras are produced by co-culturing embryos and stem cells, and keeping them physically adhered, although it may not be an assured method for producing chimeric embryos. In the injection method, the chimeras are produced by injecting stem cells into the zona pellucida using microcapillaries; however, this technique requires a high degree of skill. This study aimed to establish a novel method for producing chimeric embryos via water-in-oil droplets that differs from conventional methods. In this study, embryonic stem cells and embryos were successfully isolated in the droplets, and the emergence of chimeric embryos was confirmed by co-culture for 6 h. Using this method, the control and operability of stem cell numbers could be regulated, and reproducibility and quantification were improved during the production of chimeric embryos. In addition to the conventional methods for producing chimeric embryos, the novel method described here could be employed for the efficient production of chimeric animals.     

Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration

Emerging evidence suggests that bone marrow-derived endothelial, hematopoietic stem and progenitor cells contribute to tissue vascularization during both embryonic and postnatal physiological processes. Recent preclinical and pioneering clinical studies have shown that introduction of bone marrow-derived endothelial and hematopoietic progenitors can restore tissue vascularization after ischemic events in limbs, retina and myocardium. Corecruitment of angiocompetent hematopoietic cells delivering specific angiogenic factors facilitates incorporation of endothelial progenitor cells (EPCs) into newly sprouting blood vessels. Identification of cellular mediators and tissue-specific chemokines, which facilitate selective recruitment of bone marrow-derived stem and progenitor cells to specific organs, will open up new avenues of research to accelerate organ vascularization and regeneration. In addition, identification of factors that promote differentiation of the progenitor cells will permit functional incorporation into neo-vessels of specific tissues while diminishing potential toxicity to other organs. In this review, we discuss the clinical potential of vascular progenitor and stem cells to restore long-lasting organ vascularization and function.     

Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development.

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen 'flowing' through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.     

Stem cells. Species collage

A new study reports the first viable rat-mouse chimeras and uses rat induced pluripotent stem cells to rescue organ deficiency in mice.     

Endothelial progenitor cells for postnatal vasculogenesis

In the past decade, researchers have defined committed stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver, and reproductive organs, in both adult animals and humans. Whereas most cells in adult organs are composed of differentiated cells, which express a variety of specific phenotypic genes adapted to each organ's environment, quiescent stem or progenitor cells are maintained locally or in the systemic circulation and are activated by environmental stimuli for physiological and pathological tissue regeneration. Recently, endothelial progenitor cells (EPCs) were isolated from peripheral blood CD34, Flk-1, or AC133 antigen-positive cells, which are considered to include a hematopoietic stem cell population, and were shown to be incorporated into foci of neovascularization. This finding, that circulating EPCs may home to sites of neovascularization and differentiate into endothelial cells in situ, is consistent with "vasculogenesis," a critical paradigm for embryonic neovascularization, and suggests that vasculogenesis and angiogenesis may constitute complementary mechanisms for postnatal neovascularization. Previous reports demonstrating therapeutic potential of EPC transplantation in animal models of hindlimb and myocardial ischemia opened the way to the clinical application of cell therapy: the replacement of diseased or degenerating cell populations, tissues, and organs. In this review, we summarize biological features of EPCs and speculate on the utility of EPCs for vascular and general medicine. cell transplantation; ischemia; neovascularization; stem cell     

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells

During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4-8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

Stem cell-derived endothelial cells/progenitors migrate and pattern in the embryo using the VEGF signaling pathway

Endothelial precursor cells respond to molecular cues to migrate and assemble into embryonic blood vessels, but the signaling pathways involved in vascular patterning are not well understood. We recently showed that avian vascular patterning cues are recognized by mammalian angioblasts derived from somitic mesoderm through analysis of mouse-avian chimeras. To determine whether stem cell-derived endothelial cells/progenitors also recognize global patterning signals, murine ES cell-derived embryoid bodies (EBs) were grafted into avian hosts. ES cell-derived murine endothelial cells/progenitors migrated extensively and colonized the appropriate host vascular beds. They also formed mosaic vessels with avian endothelial cells. Unlike somite derived-endothelial cells, ES cell-derived endothelial cells/progenitors migrated across the host embryonic midline to the contralateral side. To determine the role of VEGF signaling in embryonic vascular patterning, EBs mutant for a VEGF receptor (flk-1(-/-)) or a signal (VEGF-A(-/-)) were grafted into quail hosts. Flk-1(-/-) EB grafts produced only rare endothelial cells that did not migrate or assemble into vessels. In contrast, VEGF-A(-/-) EB grafts produced endothelial cells that resembled wild-type and colonized host vascular beds, suggesting that host-derived signals can partially rescue mutant graft vascular patterning. VEGF-A(-/-) graft endothelial cells/progenitors crossed the host midline with much lower frequency than wild-type EB grafts, indicating that graft-derived VEGF compromised the midline barrier when present. Thus, ES cell-derived endothelial cells/progenitors respond appropriately to global vascular patterning cues, and they require the VEGF signaling pathway to pattern properly. Moreover, EB-avian chimeras provide an efficient way to screen mutations for vascular patterning defects.     

Generation of functional hemangioblasts from human embryonic stem cells

Recent evidence suggests the existence of progenitor cells in adult tissues that are capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Here we describe an efficient and reproducible method for generating large numbers of these bipotential progenitors-known as hemangioblasts-from human embryonic stem (hES) cells using an in vitro differentiation system. Blast cells expressed gene signatures characteristic of hemangioblasts, and could be expanded, cryopreserved and differentiated into multiple hematopoietic lineages as well as into endothelial cells. When we injected these cells into rats with diabetes or into mice with ischemia-reperfusion injury of the retina, they localized to the site of injury in the damaged vasculature and appeared to participate in repair. Injection of the cells also reduced the mortality rate after myocardial infarction and restored blood flow in hind limb ischemia in mouse models. Our data suggest that hES-derived blast cells (hES-BCs) could be important in vascular repair.     

Effects of copper deficiency on mouse yolk sac vasculature and expression of angiogenic mediators

BACKGROUND: Cu deficiency results in embryonic defects and yolk sac (YS) vasculature abnormalities. In diverse model systems, Cu treatment modulates angiogenesis, perhaps by influencing the activity of angiogenic mediators such as vascular endothelial growth factor (VEGF). Conversely, Cu chelators can suppress angiogenesis. METHODS: Gestation day (GD) 8.5 embryos from mice fed Cu-adequate (Cu+) or Cu-deficient (Cu-) diets were cultured in Cu+ or Cu- medium for 48 hr. Growth and development were evaluated, and YS vessel diameters were measured. Using RT-PCR and immunohistochemistry, the mRNA and protein expressions of VEGF, Flt-1, Flk-1, Angiopoietin-1 (Ang-1), and Tie-2 were analyzed. RESULTS: Cu+/Cu+ embryos developed normally, whereas Cu-/Cu- embryos showed a high incidence of developmental anomalies. Cu-/Cu- YS had a high proportion of vessels that were large in diameter compared to the Cu+/Cu+ YS. The mRNA expression of angiogenic mediators in Cu-/Cu- YS was similar to that in Cu+/Cu+ YS. The protein expression of VEGF in the Cu-/Cu- YS without any vessel defects, and Tie-2 in the Cu-/Cu- YS with both vessel defects and blood islands was significantly lower than that in the Cu+/Cu+ YS. The protein expression of Flt-1, Flk-1 and Ang-1 was similar among groups regardless of the presence, or type, of vessel defects. CONCLUSIONS: Results from the current study support the concept that Cu is required for the normal development of YS vasculature. Our data suggest that the impaired vascularization of Cu-deficient YS cannot be explained fully by the altered protein expression of the angiogenic growth factors reported here.     

Preimplantation mammalian aggregation and injection chimeras

The preimplantation embryo is highly resilient to experimental manipulations. A specific manipulation that has revealed many clues to the developmental process is chimera production. Chimeras have been used to describe the importance of developmental characteristics of embryonic cells and how these characteristics are involved with developmental fate. These characteristics have been monopolized in the production of interspecific chimeras and the production of transgenic animals. This review attempts to discuss the major factors affecting preimplantation mammalian embryo chimera production.     

Pdgfrb‐Cre targets lymphatic endothelial cells of both venous and non‐venous origins

The Pdgfrb‐Cre line has been used as a tool to specifically target pericytes and vascular smooth muscle cells. Recent studies showed additional targeting of cardiac and mesenteric lymphatic endothelial cells (LECs) by the Pdgfrb‐Cre transgene. In the heart, this was suggested to provide evidence for a previously unknown nonvenous source of LECs originating from yolk sac (YS) hemogenic endothelium (HemEC). Here we show that Pdgfrb‐Cre does not, however, target YS HemEC or YS‐derived erythro‐myeloid progenitors (EMPs). Instead, a high proportion of ECs in embryonic blood vessels of multiple organs, as well as venous‐derived LECs were targeted. Assessment of temporal Cre activity using the R26‐mTmG double reporter suggested recent occurrence of Pdgfrb‐Cre recombination in both blood and lymphatic ECs. It thus cannot be excluded that Pdgfrb‐Cre mediated targeting of LECs is due to de novo expression of the Pdgfrb‐Cre transgene or their previously established venous endothelial origin. Importantly, Pdgfrb‐Cre targeting of LECs does not provide evidence for YS HemEC origin of the lymphatic vasculature. Our results highlight the need for careful interpretation of lineage tracing using constitutive Cre lines that cannot discriminate active from historical expression. The early vascular targeting by the Pdgfrb‐Cre also warrants consideration for its use in studies of mural cells. genesis 54:350–358, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Activation of endothelial nitric oxide synthase is critical for erythropoietin-induced mobilization of progenitor cells

The present study aimed to define the ability of erythropoietin (EPO) to mobilize hematopoietic stem cells (c-kit(+)/sca-1(+)/lin-1(-); KSL-cells) and hematopoietic progenitor cells (CD34(+) cells), including vascular endothelial growth factor receptor 2 expressing hematopoietic progenitor cells (CD34(+)/Flk-1(+) cells). We also sought to determine the role of endothelial nitric oxide synthase (eNOS) in EPO-induced mobilization. Wild type (WT) and eNOS(-/-) mice were injected bi-weekly with recombinant erythropoietin (EPO, 1000U/kg, s.c.) for 14 days. EPO increased the number of KSL, CD34(+), CD34(+)/Flk-1(+) cells in circulating blood of wild type mice. These effects of EPO were abolished in eNOS(-/-) mice. Our results demonstrate that, EPO stimulates mobilization of hematopoietic stem and progenitor cells. This effect of EPO is critically dependent on activation of eNOS.     

Resident vascular progenitor cells: An emerging role for non-terminally differentiated vessel-resident cells in vascular biology

Throughout development and adult life the vasculature exhibits a remarkably dynamic capacity for growth and repair. The vasculature also plays a pivotal role in the execution of other diverse biologic processes, such as the provisioning of early hematopoietic stem cells during embryonic development or the regulation of vascular tone and blood pressure. Adding to this importance, from an anatomical perspective, the vasculature is clearly an omnipresent organ, with few areas of the body that it does not penetrate. Given these impressive characteristics, it is perhaps to be expected that the vasculature should require, or at least be associated with, a ready supply of stem and progenitor cells. However, somewhat surprisingly, it is only now just beginning to be broadly appreciated that the vasculature plays host to a range of vessel-resident stem and progenitor cells. The possibility that these vessel-resident cells are implicated in processes as diverse as tumor vascularization and adaptive vascular remodeling appears likely, and several exciting avenues for clinical translation are already under investigation. This review explores the various stem and progenitor cell populations that are resident in the microvasculature, endothelium, and vessel walls and vessel-resident cells capable of phenotypic transformation.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

CD117+ amniotic fluid stem cells: State of the art and future perspectives

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

CD117+ amniotic fluid stem cells

Broadly multipotent stem cells can be isolated from amniotic fluid by selection for the expression of the membrane stem cell factor receptor c-Kit, a common marker for multipotential stem cells. They have clonogenic capability and can be directed into a wide range of cell types representing the three primary embryonic lineages. Amniotic fluid stem cells maintained for over 250 population doublings retained long telomeres and a normal karyotype. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. AFS cells could be differentiate toward cardiomyogenic lineages, when co-cultured with neonatal cardiomyocytes, and have the potential to generate myogenic and hematopoietic lineages both in vitro and in vivo. Very recently first trimester AFS cells could be reprogrammed without any genetic manipulation opening new possibilities in the field of fetal/neonatal therapy and disease modeling. In this review we are aiming to summarize the knowledge on amniotic fluid stem cells and highlight the most promising results.     

Comparative angiogenic activities of induced pluripotent stem cells derived from young and old mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1(+)) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1(+) cells were sorted and shown to differentiate into VE-cadherin(+) endothelial cells and α-SMA(+) smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1(+) cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1(+) cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1(+) cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1(+) cells from young or old mice. Transplantation of Flk-1(+) cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1(+) cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging.