Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.     

A Three Dimensional Anchorage Independent In Vitro System for the Prolonged Growth of Embryoid Bodies to Study Cancer Cell Behaviour and Anticancer Agents

We describe a three dimensional (3D) anchorage independent in vitro protocol for the prolonged growth of human embryoid bodies (EBs) up to 90 days. We grew hESCs (46XX) in methylcellulose (MC) in motion culture in the presence of EB medium (EB), EB medium with Matrigel (EB + MAT), bulk culture medium (BCM), and BCM medium with Matrigel (BCM + MAT). All four experimental groups produced embryoid bodies (EBs) which with prolonged growth to 90 days acquired blood vessels and tissues from all three germ layers. Based on histology, microarray gene expression profiles and the definition for experimental teratomas, we could classify the EBs into early EBs, mature EBs and teratomas. The EB + MAT group produced the highest number of teratomas and their microarray data suggested the presence of inductive microenvironment niches and activation of pathways for self-organization, morphogenesis and growth. When we microinjected hepatocarcinoma-Green Fluorescent Protein cells (HepG2-GFP) (46XY) into the teratomas, after 10 days the HepG2-GFP cells had grown inside the teratoma as confirmed by confocal microscopy and SRY gene analysis. This 3D-MC-(EB + MAT) in vitro system requires few cells to produce many teratomas, can be used to test pluripotency of potential human embryonic and induced pluripotent stem cell lines (hESC, hiPSC), and is an experimental humanized platform to study cancer cell behavior.     

Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.     

Growth of teratomas derived from human pluripotent stem cells is influenced by the graft site

Pluripotent stem cells transplanted into immune-deficient mice form complex teratomas. Although such tumors are generally haphazard in their organization, they do contain some structures that resemble tissues normally seen in the embryo. As a consequence, the teratoma model is useful for exploring the developmental potential of stem cells and studying certain aspects of tissue development. To further our understanding of this process, we examined whether the anatomical location into which human pluripotent stem cells were grafted influenced their growth in situ. Here we report that cells grafted into the liver rapidly produced large tumors containing predominantly immature cells. In contrast, subcutaneous implants were significantly slower growing and eventually formed tumors composed of differentiated tissues. The alternative growth patterns recorded between these two graft sites indicates how environmental cues affect stem cell behavior. This approach may lead to the identification of new ways to control stem cell growth and differentiation.     

An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses. We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.     

慢病毒载体介导2次转基因对人胚胎干细胞特性的影响

目的： 研究2次转基因对人胚胎干细胞（hESCs）多能分化特点和保持自我更新能力的影响。方法：构建携带非转录因子外源基因细胞毒性T细胞相关分子4免疫球蛋白 (CTLA4Ig) 、吲哚胺2,3双氧化酶（IDO）和荧光素酶的慢病毒载体,在hESCs先转导外源基因CTLA4Ig或IDO,完成筛选后转导外源基因荧光素酶。检测hESCs中外源基因的表达和功能,免疫组化和RT-PCR以及流式细胞仪方法检测hESCs细胞表面标记物、类胚体（EB）形成和体内畸胎瘤形成能力。结果：转导的外源基因均能表达并表现相应的功能。2次转导慢病毒载体后hESCs细胞表面标记物肿瘤排斥抗原（Tra-1-60） 和Octomer转录因子-4（OCT-4）染色仍为阳性,能形成EB,RT-PCR检测到甲胎蛋白(AFP)、配对盒基因6（Pax6）和MSI1的表达,异种移植后荧光信号逐渐增强。结论：利用慢病毒2次转导非转录因子外源基因不影响hESCs的未分化状态和多能分化能力,这对hESCs的转基因研究具有借鉴价值。     

Differentiation of human embryonic stem cells after transplantation in immune-deficient mice

Our current knowledge of how human tissues grow and develop is limited. We need to increase our understanding of tissue formation if we are to fully realize the potential of stem cells as a source of material for research into health and disease and possible therapeutic applications. Transplanted pluripotent human embryonic stem cells (hESCs) provide a potential system to model and investigate cell differentiation in humans. hESCs transplanted into immune-deficient mice form complex teratomas consisting of a range of differentiated somatic tissues, some of which appear highly organized and resemble structures normally identified in the embryo and adult. Analysis of such tumors may provide a unique opportunity to study organogenesis and lead to novel approaches in bioengineering and the growth of functioning structures composed of a range of alternative cell types. However, little has been done to characterize the developmental potential of hESCs after transplantation. This concise review presents evidence for the ability of hESCs to differentiate in vivo and highlights some of the prominent questions that need to be addressed if transplantation is to be used as a research tool to study hESC differentiation.     

Spatial and temporal kinetics of teratoma formation from murine embryonic stem cell transplantation

Pluripotent embryonic stem (ES) cells have the potential to form teratomas composed of derivatives from all three germ layers in animal models. This tumorigenic potential prevents clinical translation of ES cell research. In order to understand the biology and physiology of teratoma formation, we investigated the influence of undifferentiated ES cell number, migration, and long-term follow up after transplantation. Murine ES cells were stably transduced with a self-inactivating (SIN) lentiviral vector with a constitutive ubiquitin promoter driving a double-fusion (DF) reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP). To assess effects of cell numbers, varying numbers of ES-DF cells (1, 10, 100, 1,000, and 10,000) were injected subcutaneously into the dorsal regions of adult nude mice. To assess cell migration, 1 x 10(6) ES-DF cells were injected intramyocardially into adult Sv129 mice, and leakage to other extracardiac sites was monitored. To assess effects of long-term engraftment, 1 x 10(4) ES-DF cells were injected intramyocardially into adult nude rats, and cell survival response was monitored for 10 months. Our results show that ES-DF cells caused extracardiac teratoma in both immunocompetent and immunodeficient hosts; the lowest number of undifferentiated ES cells capable of causing teratoma was 500-1,000; and long-term engraftment could be shown for >300 days. Collectively, these results illustrate the potent tumorigenic potential of ES cells, which presents an enormous obstacle for future clinical studies.     

Is gliomatosis peritonei derived from the associated ovarian teratoma?

Gliomatosis peritonei, a rare condition that occurs almost exclusively in the setting of ovarian immature teratoma, is characterized by the occurrence of nodules of mature glial tissues in the peritoneum. It is controversial whether glial tissues are derived from maturation of the associated teratomatous tissue that has implanted in the peritoneum, or glial differentiation of subperitoneal stem cells. In this study, we employed the unique genetic characteristics of ovarian teratomas (often with a duplicated set of maternal chromosomes and thus homozygous at many polymorphic microsatellite loci) versus normal tissues (heterozygous pattern due to presence of maternal and paternal genetic materials) to investigate the origin of gliomatosis peritonei. DNA samples were extracted from microdissected paraffin-embedded tissues, including the glial implants, the associated ovarian teratomas, and normal tissues, to determine their patterns of microsatellite loci in a multiplex polymerase chain reaction system. Two cases were not informative because the ovarian teratoma showed a heterozygous microsatellite pattern. In the 5 informative cases, the normal tissues showed a heterozygous pattern in the microsatellite loci, the associated teratomas showed a homozygous pattern, and the glial tissues showed a heterozygous pattern. Thus, gliomatosis peritonei is genetically unrelated to the associated teratoma but is probably derived from nonteratomatous cells, such as through metaplasia of submesothelial cells.     

Induced Pluripotent Stem Cells from Human Hair Follicle Mesenchymal Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.     

Effect of etoposide on experimental testicular teratoma in 129/SvJ mice

To study the effects of etoposide on experimental testicular teratoma in 129/SvJ mouse we analysed the tumour growth, differentiation, apoptosis and the localisation of mdr₁ P-glycoprotein (mdr₁-Pgp). In this model the implanted gonadal ridges developed into testicular teratomas in 17 out of 56 implanted testes (30%) and in 14 out of 28 mice (50%). The tumour-bearing mice were treated with etoposide on 4 successive days either 4 weeks or 6 weeks after implantation, and killed 7 days after the last dose. The mice in the control groups did not receive etoposide. The teratomas consisted mainly of neural tissue. The etoposide-treated 4-week teratomas, but not the 6-week teratomas, were significantly smaller than those in the corresponding control groups. The density of apoptotic cells and the distribution of the mdr₁-Pgp were not altered by etoposide. The decreased proportion of immature neuroectodermal tissue components was observed in all treated teratomas, converting the histology towards that of a mature teratoma. In addition, a low proportion of immature tissue components was frequently combined with a low density of apoptotic cells. In conclusion, etoposide decreased the immature tissue components of teratomas, while mature tissues remained unaffected. These results may have clinical relevance in man, since they confirm that postchemotherapy mature teratomas cannot be treated with chemotherapy. Despite benign histology, the human residual tumours have a significant malignant potential and require complete surgical excision and close surveillance. Received: 20 August 1999 / Accepted: 20 January 2000     

Human very Small Embryonic-like Cells Support Vascular Maturation and Therapeutic Revascularization Induced by Endothelial Progenitor Cells

Very small embryonic-like stem cells (VSELs) are major pluripotent stem cells defined as cells of small size being Lineage- negative, CD133-positive, and CD45-negative. We previously described that human bone marrow VSELs were able to differentiate into endothelial cells and promoted post-ischemic revascularization in mice with surgically induced critical limb ischemia. In the present work, we isolated bone marrow VSELs from patients with critical limb ischemia and studied their ability to support endothelial progenitor cells therapeutic capacity and revascularization potential. Sorted bone marrow VSELs cultured in angiogenic media were co-injected with endothelial progenitor cells and have been show to trigger post-ischemic revascularization in immunodeficient mice, and support vessel formation in vivo in Matrigel implants better than human bone marrow mesenchymal stem cells. In conclusion, VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial and perivascular lineage in humans. VSELs are the first real vasculogenic stem cells able to differentiate in endothelial and perivascular lineage in human adult described from now. Thus, because VSELs presence have been proposed in adult tissues, we think that VSELs are CD45 negative stem cells able to give rise to vascular regeneration in human tissues and vessels.     

The osteoinductive potential of printable, cell-laden hydrogel-ceramic composites

Hydrogels used as injectables or in organ printing often lack the appropriate stimuli to direct osteogenic differentiation of embedded multipotent stromal cells (MSCs), resulting in limited bone formation in these matrices. Addition of calcium phosphate (CaP) particles to the printing mixture is hypothesized to overcome this drawback. In this study we have investigated the effect of CaP particles on the osteoinductive potential of cell-laden hydrogel-CaP composite matrices. To this end, apatitic nanoparticles have been included in Matrigel constructs where after the viability of embedded progenitor cells was assessed in vitro. In addition, the osteoinductive potential of cell-laden Matrigel containing apatitic nanoparticles was investigated in vivo and compared with composites containing osteoinductive biphasic calcium phosphate (BCP) microparticles after subcutaneous implantation in immunodeficient mice. Histological and immunohistochemical analysis of the tissue response as well as in vivo bone formation revealed that apatitic nanoparticles were osteoinductive and induced osteoclast activation, but without bone formation. The BCP particles were more effective in inducing elaborate bone formation at the ectopic location.     

Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.     

Human-serum matrix supports undifferentiated growth of human embryonic stem cells

One of the most frequently used matrices for feeder-free growth of undifferentiated human embryonic stem cells (hESCs) is Matrigel, which supports attachment and growth of undifferentiated hESCs in the presence of mouse embryonic fibroblast-conditioned medium. Unfortunately, application of Matrigel or medium conditioned by mouse embryonic feeder cells is not ideal for potential medical application of hESCs because xenogeneic pathogens can be transmitted through culture conditions. We demonstrate here that human serum as matrix and medium conditioned by differentiated hESCs reduce exposure of hESCs to animal ingredients and provide a safer direction toward completely animal-free conditions for application, handling, and understanding of hESC biology. At the same time, hESCs grown under these conditions maintain all hESC features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype.     

Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation

Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinson's disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC-derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion-induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.