Preferential Hematopoietic Differentiation in Induced Pluripotent Stem Cells Derived From Human Umbilical Cord Arterial Endothelial Cells.

BACKGROUND: The major obstacle for applications of human induced pluripotent stem cells (hiPSCs) is efficient and controlled lineage-specific differentiation. Hence, a deeper understanding of the initial populations of hiPSCs is required to instruct proficient lineage commitment. METHODS: hiPSCs were generated from somatic cells by transduction of 4 human transcription factors (OCT4, SOX2, KLF4, and C-MYC) using Sendai virus vectors. Genome-wide DNA methylation analysis and transcriptional analysis were performed to evaluate the pluripotent capacity and somatic memory state of hiPSCs. Flow cytometric analysis and colony assays were performed to assess the hematopoietic differentiation capacity of hiPSCs. RESULTS: Here, we reveal human umbilical arterial endothelial cell-derived induced pluripotent stem cells (HuA-iPSCs) exhibit indistinguishable pluripotency in comparison with human embryonic stem cells and hiPSCs derived from other tissues of origin (umbilical vein endothelial cells, cord blood, foreskin fibroblasts, and fetal skin fibroblasts). However, HuA-iPSCs retain a transcriptional memory typical of the parental human umbilical cord arterial endothelial cells, together with a strikingly similar DNA methylation signature to umbilical cord blood-derived induced pluripotent stem cells that distinguishes them from other human pluripotent stem cells. Ultimately, HuA-iPSCs are most efficient in targeted differentiation toward hematopoietic lineage among all human pluripotent stem cells based on the functional and quantitative evaluation of both flow cytometric analysis and colony assays. Application of the Rho-kinase activator significantly reduces the effects of preferential hematopoietic differentiation in HuA-iPSCs, reflected in CD34+ cell percentage of day 7, hematopoietic/endothelial-associated gene expression, and even colony-forming unit numbers. CONCLUSIONS: Collectively, our data suggest that somatic cell memory may predispose HuA-iPSCs to differentiate more amenably into hematopoietic fate, bringing us closer to generating hematopoietic cell types in vitro from nonhematopoietic tissue for therapeutic applications.

Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells.

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38- and CD34+ CD38-CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

HGF Mediates Clinical-Grade Human Umbilical Cord-Derived Mesenchymal Stem Cells Improved Functional Recovery in a Senescence-Accelerated Mouse Model of Alzheimer's Disease.

Stem cells have emerged as a potential therapy for a range of neural insults, but their application in Alzheimer's disease (AD) is still limited and the mechanisms underlying the cognitive benefits of stem cells remain to be elucidated. Here, the effects of clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the recovery of cognitive ability in SAMP8 mice, a senescence-accelerated mouse model of AD is explored. A functional assay identifies that the core functional factor hepatocyte growth factor (HGF) secreted from hUC-MSCs plays critical roles in hUC-MSC-modulated recovery of damaged neural cells by down-regulating hyperphosphorylated tau, reversing spine loss, and promoting synaptic plasticity in an AD cell model. Mechanistically, structural and functional recovery, as well as cognitive enhancements elicited by exposure to hUC-MSCs, are at least partially mediated by HGF in the AD hippocampus through the activation of the cMet-AKT-GSK3ß signaling pathway. Taken together, these data strongly implicate HGF in mediating hUC-MSC-induced improvements in functional recovery in AD models.

Arterial endothelium creates a permissive niche for expansion of human cord blood hematopoietic stem and progenitor cells.

BACKGROUND: Although cord blood (CB) offers promise for treatment of patients with high-risk hematological malignancies and immune disorders, the limited numbers of hematopoietic stem cell (HSC)/progenitor cell in a CB unit and straitened circumstances in expanding ex vivo make it quite challenging to develop the successful cell therapies. METHODS: In this study, a novel strategy has been developed to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) by coculture with engineered human umbilical arterial endothelial cells (HuAECs-E4orf1-GFP), which expresses E4ORF1 stably by using a retroviral system. RESULTS: Coculture of CD34+ hCB cells with HuAECs-E4orf1-GFP resulted in generation of considerably more total nucleated cells, CD34+CD38-, and CD34+CD38-CD90+ HSPCs in comparison with that of cytokines alone or that of coculture with human umbilical vein endothelial cells (HuVECs) after 14-day amplification. The in vitro multilineage differentiation potential and in vivo repopulating capacity of the expanded hematopoietic cells cocultured with HuAECs-E4orf1-GFP were also markedly enhanced compared with the other two control groups. DLL4, a major determinant of arterial endothelial cell (EC) identity, was associated with CD34+ hCB cells amplified on HuAECs-E4orf1-GFP. CONCLUSIONS: Collectively, we demonstrated that HuAECs acted as a permissive niche in facilitating expansion of HSPCs. Our study further implicated that the crucial factors and related pathways presented in HuAECs may give a hint to maintain self-renewal of bona fide HSCs.

[Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro].

OBJECTIVE: To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. METHODS: hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. RESULTS: The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. CONCLUSION: hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

Integration-free reprogramming of human umbilical arterial endothelial cells into induced pluripotent stem cells IHSTMi001-A.

Primary arterial endothelial cell (AEC) is an attractive source of tissue-engineered blood vessels for therapeutic transplantation in vascular disease. However, scarcity of donor tissue, inability of proliferation and undergo de-differentiation in culture remain major obstacles. We derived a stable induced pluripotent stem cell (iPSC) line possessed all the characteristics of pluripotent state from human umbilical arterial endothelial cells by transduction of four human transcription factors (Oct4, Sox2, Klf4, and c-Myc) using sendai virus vectors. It will likely facilitate to lineage differentiate and generate sufficient AECs for clinical use in cardiovascular disease based on epigenetic memory of the tissue of origin.

Clinical-grade human umbilical cord-derived mesenchymal stem cells reverse cognitive aging via improving synaptic plasticity and endogenous neurogenesis.

Cognitive aging is a leading public health concern with the increasing aging population, but there is still lack of specific interventions directed against it. Recent studies have shown that cognitive function is intimately affected by systemic milieu in aging brain, and improvement of systemic environment in aging brain may be a promising approach for rejuvenating cognitive aging. Here, we sought to study the intervention effects of clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on cognitive aging in a murine model of aging. The conventional aging model in mice induced by d-galactose (d-gal) was employed here. Mice received once every two weeks intraperitoneal administration of hUC-MSCs. After 3 months of systematical regulation of hUC-MSCs, the hippocampal-dependent learning and memory ability was effectively improved in aged mice, and the synaptic plasticity was remarkably enhanced in CA1 area of the aged hippocampus; moreover, the neurobiological substrates that could impact on the function of hippocampal circuits were recovered in the aged hippocampus reflecting in: dendritic spine density enhanced, neural sheath and cytoskeleton restored, and postsynaptic density area increased. In addition, the activation of the endogenic neurogenesis which is beneficial to stabilize the neural network in hippocampus was observed after hUC-MSCs transplantation. Furthermore, we demonstrated that beneficial effects of systematical regulation of hUC-MSCs could be mediated by activation of mitogen-activated protein kinase (MAPK)-ERK-CREB signaling pathway in the aged hippocampus. Our study provides the first evidence that hUC-MSCs, which have the capacity of systematically regulating the aging brain, may be a potential intervention for cognitive aging.

Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes.

BACKGROUND: Bi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases. METHODS: In this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium. RESULTS: In vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression. CONCLUSIONS: Here we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

Generation of induced pluripotent stem cells from Chinese hamster embryonic fibroblasts.

We derived a stable cell line from Chinese hamster embryonic fibroblasts by transduction of four mouse transcription factors (M3O, Sox2, Klf4, and n-Myc) using a lentiviral vector. The cell line possess all the characteristics of an induced pluripotent stem cell (iPSC) line. Given that Chinese hamster ovary (CHO) cells are the predominant host cells used for therapeutic protein production and no pluripotent stem cell line or other normal cell line has been isolated from Chinese hamster, this iPSC line may serve as a useful tool for research using CHO cells or even be used for deriving new cell lines.

Hepatogenesis of adipose-derived stem cells on poly-lactide-co-glycolide scaffolds: in vitro and in vivo studies.

Human adipose-derived stem cells (hASCs) have been shown to be multipotent and could be induced into various cell types, which make them the ideal cell source for cell therapy or tissue engineering. However, differentiation of ASCs into hepatocytes on three-dimensional scaffold, an important part of tissue engineering, has not been reported. In this study, to investigate the hepatogenesis of ASCs on porous poly-lactide-co-glycolide (PLGA) scaffolds, we loaded hASCs on these scaffolds. The cell-scaffold complex was implanted into the peritoneal cavity of 70% hepatectomized rats with or without 14 days of induction in hepatic inducing medium. Our results indicated that hASCs cultured on the PLGA scaffolds in the hepatic inducing medium proliferated more efficiently and could be induced into cells with hepatocyte-like phenotypic and functional properties. In vivo studies showed that induced hASCs on PLGA scaffolds survived and maintained hepatic phenotype and function for at least 14 days after implantation; moreover, noninduced hASCs on PLGA scaffolds expressed human albumin 14 days after transplantation. Collectively, these results suggest that porous PLGA scaffolds are suitable for the hepatogenesis of hASCs. These findings might be helpful in the application of hASC-based tissue engineering for liver disease therapy.

Lineage restriction and differentiation of human embryonic stem cells into hepatic progenitors and zone 1 hepatocytes.

Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

[Lyophilization for platelet preservation].

To explore a new lyophilized preservation methods for human platelets, platelets were pre-treated with aldehyde, human albumin or trehalose was added to the system of condensed cooling as protectant to stabilize the structure of platelets. The optimal resuspending buffer was also selected in the study. The morphological changes of platelets were observed by using electron microscopy after lyophilization, and the expression of membrane proteins on platelets was detected also after lyophilization. The results indicated that the recovery rate of platelets treated with aldehyde was generally more than 60%. Aggregative ability was reduced a little than the platelet untreated. 5% of human albumin had an advantage over 40 mmol/L of trehalose in respect of the preservation effect. In the way of keeping aggregative ability, PPP was obviously better than PBS. The results of electron microscopy displayed that organelles including mitochondria and excreted granules could be observed distinctly. Whereas, expression of membrane proteins of platelet treated with aldehyde was evidently dropped as compared with those of the fresh platelet. In conclusion, aldehyde as a novel protective agent, has excellent effects on lyophilization of platelets and is worthy to be further studied.