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Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single‐cell resolution. Interestingly, we detected a few progenitor cells and non‐erythroid cells from both origins. In PSC‐derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN‐derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic‐related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony‐forming unit‐erythroid cells. Inhibition of CD99 reduced the proliferation of PSC‐derived cells and facilitated erythroid maturation. Furthermore, CD99–CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation. [ABSTRACT FROM AUTHOR]

Derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom(+) hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of the tdTom(+) population. Notably, RUNX1c/tdTom(+) cells represent only a limited subpopulation of the CD34(+) CD45(+) and CD34(+) CD43(+) cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom(+) cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom(+) population to CD34(+) CD45(+) umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs.

Biological scaffolds based stem cell delivery methods have emerged as a promising approach for tissue repair and regeneration. Here we developed a hydrogel biological scaffold from human decellularized adipose matrix (hDAM) for human adipose‐derived stem cells (hASCs) delivery to accelerate chronic wound healing. The hDAM hydrogel was prepared by pepsin mediated digestion and pH controlled neutralization. The morphology, survival, proliferation, and angiogenic paracrine activity of hASCs cultured in the hydrogel were assessed. Moreover, the therapeutic efficacy of the hASCs‐hydrogel composite for impaired wound healing was evaluated by using a full‐thickness wound model on diabetic mouse. The developed hDAM hydrogel was a thermosensitive hydrogel, presented the biochemical complexity of native extracellular matrix and formed a porous nanofiber structure after gelation. The hydrogel can support hASCs adhesion, survival, and proliferation. Compared to standard culture condition, hASCs cultured in the hydrogel exhibited enhanced paracrine activity with increased secretion of hepatocyte growth factor. In the diabetic mice model with excisional full‐thickness skin wounds, mice treated with the hASCs‐hydrogel composite displayed accelerated wound closure and increased neovascularization. Our results suggested that the developed hDAM hydrogel can provide a favorable microenvironment for hASCs with augmented regeneration potential to accelerate chronic wound healing. [ABSTRACT FROM AUTHOR]

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 10(9)-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive ß -globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion. [ABSTRACT FROM AUTHOR]

The accurate control of early cell fate specification during differentiation of human embryonic stem cells (hESCs) is critical for acquiring pure therapeutic cell populations of interest. Bone morphogenetic protein 4 (BMP4) is a key mesoderm inducer from ESCs. However, the molecular mechanism of the mesodermal cell fate decision induced by BMP4 remains unclear. Here, we demonstrate the requirement of a bioactive lipid, prostaglandin E 2 (PGE 2 ), for the mesoderm specification from hESCs by BMP4 induction. We show that BMP4 directly regulates the expression of the key enzyme for PGE 2 synthesis, COX-1, and promotes PGE 2 production. More importantly, in the absence of BMP4, forced COX-1 expression or PGE 2 treatment is sufficient to initiate mesoderm specification of hESCs by activation of EP2-PKA signaling and modulation of nuclear translocation of β-catenin. Together, our findings provide insights into the critical role of BMP regulation of PGE 2 synthesis and its downstream signaling in initiating mesoderm commitment of hESCs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the co-transplantation of C57-green fluorescent protein (GFP) mouse epidermis and dermis cells subcutaneously to induce the hair follicle regeneration., Methods: C57-GFP mouse epidermis and dermis were harvested for isolation the mouse epidermis and dermis cells. The morphology of epidermis and dermis mixed cells at ratio of 1:1 of adult mouse, dermis cells of adult mouse, cultured 3rd generation dermis cells were observed by fluorescence microscope. Immunocytochemistry staining was used to detect hair follicle stem cells markers in cultured 3rd generation dermis cells from new born C57-GFP mouse. And then the epidermis and dermis mixed cells of adult mouse (group A), dermis cells of adult mouse (group B), cultured 3rd generation dermis cells of new born mouse (group C), and saline (group D) were transplanted subcutaneously into Balb/c nude mice. The skin surface of nude mice were observed at 4, 5, 6 weeks of transplantation and hair follicle formation were detected at 6 weeks by immunohistochemistry staining., Results: The isolated C57-GFP mouse epidermis and dermis cells strongly expressed the GFP under the fluorescence microscope. Immunocytochemistry staining for hair follicle stem cells markers in cultured 3rd generation dermis cells showed strong expression of Vimentin and α-smooth muscle actin, indicating that the cells were dermal sheath cells; some cells expressed CD133, Versican, and cytokeratin 15. After transplanted for 4-6 weeks, the skin became black at the injection site in group A, indicating new hair follicle formation. However, no color change was observed in groups B, C, and D. Immunohistochemical staining showed that new complete hair follicles structures formed in group A. GFP expression could be only observed in the hair follicle dermal sheath and outer root sheath in group B, and it could also be observed in the hair follicle dermal sheath, outer root sheath, dermal papilla cells, and sweat gland in group C. The expression of GFP was negative in group D., Conclusion: Co-transplantation of mouse epidermis and dermis cells can induce the hair follicle regeneration by means of interaction of each other. And transplantation of isolated dermis cells or cultured dermis cells individually only partly involved in the hair follicles formation.

Objective: To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs)., Methods: UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing., Results: With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective., Conclusions: This non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Objective: To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells., Methods: Red blood cells were precipitated by hydroxyethyl starch (HES). Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed., Results: The best choice for erythrocyte sedimentation and high efficiency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with 116t (IL-11, IL-6, TPO), st36(SCF, TPO, IL-3, IL-6), pt36 (PDGF,TPO,IL-3,IL-6) or pst36 for 7 days. St36 group (50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6) yielded the most CD41/CD61 positive [(6.79±1.97)×10⁴]. The cell viability [(82.85 ± 0.64)%] of st36 group by using of imported serum-free medium was better than [(60.90±6.93)%] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [(18.60±1.97)×10⁴] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached (54.27 ± 6.31)% on day 14. Wright-Giemsa staining showed that different phase cells, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days'culture. Clone forming unit-megakarocytes (CFU-MK) assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [1 236.0±32.9] on day 14, which was higher than that in medium without induction (P<0.01). Platelets from megakaryocytes showed agglutination function after 10 days'culture., Conclusion: 1.5% HES was the best solution to precipitate erythrocytes. The combination of an imported serum-free medium with IL-3, IL-6, SCF and TPO showed better induction efficiency than domestic medium or other cytokine cocktails. Meanwhile, induced megakaryocytes produced functional platelets.

The presence of mesenchymal progenitor cells within bone marrow has been known since the late nineteenth century. To date, mesenchymal stem cells (MSCs) have been isolated from several different connective tissues, such as adipose tissue, muscle, placenta, umbilical cord matrix, blood, liver, and dental pulp. Bone marrow, however, is still one of the major sources of MSCs for preclinical and clinical research. MSCs were first evaluated for regenerative applications and have since been shown to directly influence the immune system and to promote neovascularization of ischemic tissues. These observations have prompted a new era of MSC transplantation as a treatment for various diseases. In this review, we summarize the important studies that have investigated the use of MSCs as a therapeutic agent for regenerative medicine, immune disorders, cancer, and gene therapy. Furthermore, we discuss the mechanisms involved in MSC-based therapies and clinical-grade MSC manufacturing.