Transient PU.1 low fetal progenitors generate lymphoid progeny that contribute to adult immunity.

Hematopoietic stem cells and multipotential progenitors emerge in multiple, overlapping waves of fetal development. Some of these populations seed the bone marrow and sustain adult B- and T-cell development long-term after birth. However, others are present transiently, but whether they are vestigial or generate B and T cells that contribute to the adult immune system is not well understood. We now report that transient fetal progenitors distinguished by expression of low levels of the PU.1 transcription factor generated activated and memory T and B cells that colonized and were maintained in secondary lymphoid tissues. These included the small and large intestines, where they may contribute to the maintenance of gut homeostasis through at least middle age. At least some of the activated/memory cells may have been the progeny of B-1 and marginal zone B cells, as transient PU.1low fetal progenitors efficiently generated those populations. Taken together, our data demonstrate the potential of B- and T-cell progeny of transient PU.1low fetal progenitors to make an early and long-term contribution to the adult immune system.

Transient characteristics of universal cells on human-induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources.

INTRODUCTION: It is important to prepare 'hypoimmunogenic' or 'universal' human pluripotent stem cells (hPSCs) with gene-editing technology by knocking out or in immune-related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off-the-shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs. METHODS: Universal human-induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2-5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)-expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions. RESULTS: Our universal hiPSCs during passages 10-25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21-22 survived and continued beating even after treatment with allogenic mononuclear cells.

Identification of hepatic progenitor cells in the canine fetal liver.

The liver plays essential roles in human and animal organisms, such as the storage, release, metabolism, and elimination of various endogenous or exogenous substances. Although its vital importance, few treatments are yet available when a hepatic failure occurs, and hence, the use of stem cells has arisen as a possible solution for both human and veterinary medicines. Previous studies have shown the existence of hepatic progenitor cells in human fetuses that were positive for EpCAM and NCAM. There is limited evidence, however, further identification and characterization of these cells in other species. Considering the similarity between dogs and humans regarding physiology, and also the increasing importance of developing new treatments for both veterinary and translational medicine, this study attempted to identify hepatic progenitor cells in canine fetal liver. For that, livers from canine fetuses were collected, cells were isolated by enzymatic digestion and cultured. Cells were characterized regarding morphology and expression of EpCAM, NCAM, Nestin, and Thy-1/CD90 markers. Our results suggest that it is possible to identify hepatic progenitor cells in the canine fetal liver; however, for therapeutic use, further techniques for cellular isolation and culture are necessary to obtain enriched populations of hepatic progenitors from the canine fetal liver.

Direct Reprogramming of Human Fetal- and Stem Cell-Derived Glial Progenitor Cells into Midbrain Dopaminergic Neurons.

Human glial progenitor cells (hGPCs) are promising cellular substrates to explore for the in situ production of new neurons for brain repair. Proof of concept for direct neuronal reprogramming of glial progenitors has been obtained in mouse models in vivo, but conversion using human cells has not yet been demonstrated. Such studies have been difficult to perform since hGPCs are born late during human fetal development, with limited accessibility for in vitro culture. In this study, we show proof of concept of hGPC conversion using fetal cells and also establish a renewable and reproducible stem cell-based hGPC system for direct neural conversion in vitro. Using this system, we have identified optimal combinations of fate determinants for the efficient dopaminergic (DA) conversion of hGPCs, thereby yielding a therapeutically relevant cell type that selectively degenerates in Parkinson's disease. The induced DA neurons show a progressive, subtype-specific phenotypic maturation and acquire functional electrophysiological properties indicative of DA phenotype.

Generation of foxn1/Casper Mutant Zebrafish for Allograft and Xenograft of Normal and Malignant Cells.

Cell transplantation into immunodeficient recipients is a widely used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a foxn1/Casper mutant zebrafish that is transparent and exhibits T cell deficiency. By employing the line for hematopoietic stem cell (HSC) transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization zebrafish embryos produce a better transplant outcome in foxn1/Casper mutants, compared with adult HSCs. In addition to HSCT, the foxn1/Casper mutant is feasible for allografts of myelodysplastic syndrome-like and muscle cells, as well as xenografts of medaka muscle cells. In summary, foxn1/Casper mutants permit the nonconditioned engraftment of multiple cell types and visualized characterization of transplanted cells in vivo.

Direct effects of transforming growth factor-ß1 signaling on the differentiation fate of fetal hepatic progenitor cells.

Aim: To investigate direct roles of TGF-ß1 signaling in the differentiation process of fetal hepatic progenitor cells (HPCs). Materials & methods: Exogenous TGF-ß1 and SB431542 were added into fetal HPCs. Then, SB431542 was intraperitoneally injected into pregnant mice for 8 days. Results: Fetal HPCs treated with TGF-ß1 differentiated into cholangiocytes. However, hepatocyte marker was highly expressed after inhibiting TGF-ß1 signaling. In vivo, hematopoietic cells were gradually replaced with liver cells and TGF-ß1 expression was evidently decreased as fetal liver developed. Inhibition of TGF-ß1 signaling caused increase of ALB+ cells, but CK19 expression was more obvious in control mice livers. Conclusion: TGF-ß1 signaling may play decisive roles in fetal HPCs differentiation into functional hepatocytes or cholangiocytes.

Direct Lineage Reprogramming of Mouse Fibroblasts to Acquire the Identity of Fetal Intestine-Derived Progenitor Cells.

Intestinal organoids are useful models for studying the characteristics of intestinal diseases and their treatment. However, a major limiting factor in their usability is the need for donor tissue fragments or pluripotent stem cells to generate the organoids. Here, we describe an approach to generate intestinal organoids from fibroblasts, a new source. We used direct reprogramming technology to generate cells with the properties of fetal intestine-derived progenitor cells (FIPCs) from mouse embryonic fibroblasts (MEFs). These induced FIPCs (iFIPCs) can give rise to cells resembling intestinal stem cells (ISCs), henceforth referred to as induced ISCs (iISCs). These iFIPCs and iISCs form spherical and budding organoids, respectively, similar to FIPCs and ISCs. These induced intestinal organoids could be used for studies on intestinal diseases and regenerative therapy.

Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2.

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

Engineered cartilage utilizing fetal cartilage-derived progenitor cells for cartilage repair.

The aim of this study was to develop a fetal cartilage-derived progenitor cell (FCPC) based cartilage gel through self-assembly for cartilage repair surgery, with clinically useful properties including adhesiveness, plasticity, and continued chondrogenic remodeling after transplantation. Characterization of the gels according to in vitro self-assembly period resulted in increased chondrogenic features over time. Adhesion strength of the cartilage gels were significantly higher compared to alginate gel, with the 2-wk group showing a near 20-fold higher strength (1.8 ± 0.15 kPa vs. 0.09 ± 0.01 kPa, p < 0.001). The in vivo remodeling process analysis of the 2 wk cultured gels showed increased cartilage repair characteristics and stiffness over time, with higher integration-failure stress compared to osteochondral autograft controls at 4 weeks (p < 0.01). In the nonhuman primate investigation, cartilage repair scores were significantly better in the gel group compared to defects alone after 24 weeks (p < 0.001). Cell distribution analysis at 24 weeks showed that human cells remained within the transplanted defects only. A self-assembled, FCPC-based cartilage gel showed chondrogenic repair potential as well as adhesive properties, beneficial for cartilage repair.

Effect of Transplantation of Neural Stem and Progenitor Cells on Memory in Animals with Alzheimer's Type Neurodegeneration.

The effects of systemic and intracerebral transplantation of human fetal neural stem and progenitor cells were studied on the model of olfactory bulbectomy in mice with developing signs of sporadic Alzheimer's disease. It was found that transplantation of these cells at certain stages of disease development contributed to improvement of spatial memory and preservation of hippocampal neurons in these animals.

Human Chorionic Gonadotropin Improves the Proliferation and Regenerative Potential of Bone Marrow Adherent Stem Cells and the Immune Tolerance of Fetal Microchimeric Stem Cells In Vitro.

Nongonadal tissues express luteinizing hormone-chorionic gonadotropin receptors (LHCG-R) which are essential for their growth during fetal development. Adult mesenchymal stem/stromal cells (MSCs) have been shown to express functional LHCG-R outside pregnancy conditions, making them susceptible to hCG stimulation. In the present study we tested the effect of hCG treatment on bone marrow (BM) derived adherent stem cells in vitro, isolated from a parous women, mother of male sons, in order to evaluate its effect on maternal MSCs and in the same time on fetal microchimeric stem cells (FMSCs), to better understand the outcomes of this safe and affordable treatment on cell proliferation and expression of pluripotency genes. Our study highlights the beneficial effects of hCG exposure on gene regulation in bone marrow adherent stem cells through the upregulation of pluripotency genes and selection of more primitive mesenchymal stem cells with a better differentiation potential. Validation of these effects on MSCs and FMSCs long after parturition in vivo represents a close perspective as it could set the premises of a new mobilization strategy for the stem cell transplantation procedures in the clinical setting.

New therapeutic target for pediatric anaplastic ependymoma control: study of anti-tumor activity by a Kunitz-type molecule, Amblyomin-X.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

Isolation and Characterization of a Fetal-Maternal Microchimeric Stem Cell Population in Maternal Hair Follicles Long after Parturition.

Fetal-maternal microchimerism describes the acquisition of fetal stem cells (FSC) by the mother during pregnancy and their long-term persistence after parturition. FSC may engraft in a variety of maternal tissues especially if there is organ/tissue injury, but their role and mechanism of persistence still remains elusive. Clinical applications due to their pluripotency, immunomodulatory effects and accessibility make them good candidates for ex-vivo manipulation and autologous therapies. The hair follicles contain a distinctive niche for pluripotent stem cells (PSC). To date, there is no published evidence of fetal microchimerism in the hair follicle. In our study, follicular unit extraction (FUE) technique allowed easy stem cell cultures to be obtained while simple hair follicle removal by pull-out technique failed to generate stem cells in culture. We identified microchimeric fetal stem cells within the primitive population of maternal stem cells isolated from the hair follicles with typical mesenchymal phenotype, expression of PSC genes and differentiation potential towards osteocytes, adypocites and chondrocytes. This is the first study to isolate fetal microchimeric stem cells in adult human hair long after parturition. We presume a sanctuary partition mechanism with PSC of the mother deposited during early embryogenesis could explain their long-term persistence.

In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

Immunophenotype and Immune-Modulatory Activities of Human Fetal Cartilage-Derived Progenitor Cells.

We have previously reported human fetal cartilage progenitor cells (hFCPCs) as a novel source of therapeutic cells showing high proliferation and stem cell properties superior to those of adult mesenchymal stem cells (MSCs). In this study, we investigated the immunophenotype and immune-modulatory activities of hFCPCs. With institutional review board approval, hFCPCs were isolated from fetuses at 11-13 weeks of gestation. hFCPCs showed strong expression of HLA class I molecules but low or no expression of HLA class II and co-stimulatory molecules, which was not changed significantly after 4 days of IFN-γ treatment. In a mixed lymphocyte reaction (MLR), hFCPCs showed no allogeneic immune response to peripheral blood lymphocytes (PBLs) and suppressed concanavalin A (Con A)-mediated proliferation of PBLs in a dose-dependent manner. In addition, hFCPCs inhibited Con A-induced secretion of pro-inflammatory cytokines TNF-α and IFN-γ from PBLs but showed no significant decrease of secretion of IL-10, anti-inflammatory cytokine. Co-culture of hFCPCs with stimulated PBLs for 4 days resulted in a significant increase in CD4+CD25+FoxP3+ T regulatory cells (Tregs). hFCPCs expressed LIF, TGF-ß1, TSG-6, and sHLA-G5 but did not express IDO and HGF. Stimulation of hFCPCs with TNF-α for 12 h showed slight induction in the expression of LIF, TSG-6, IDO, and HGF, whereas stimulation with IFN-γ did not affect expression of any of these factors. These results suggest that hFCPCs have low allogeneic immunogenicity and immune-modulatory activity in vitro, comparable to those of MSCs. However, compared with MSCs, hFCPCs were less responsive to TNF-α and IFN-γ, and the mechanisms underlying responses to these two cell types appeared distinct.

In vitro artificial skin engineering by decellularized placental scaffold for secondary skin problems of meningomyelocele.

BACKGROUND: Meningomyelocele (MMC) is a condition that is originated by the fusion defect of the neural tube. It is a congenital anomaly and can be characterized by spinal cord defects and impaired skin integrity. It is very important to close the skin openings via three-dimensional artificial skin like construction for preventing infection and maintaining the healthy skin structure. Therefore, we aim to generate artificial skin like structures formed by the own cells of donor for treating the MMC-related skin disorder. METHODS: In this study, waste placental tissues were collected and decellularization process was applied. Decellularized and normal placental tissues were compared by immunohistochemistry (IHC). Donor's own placental stem cells were seeded onto biological scaffold and were differentiated into skin related cell types. Finally, gene expressions were evaluated, and the structural integrity were analyzed with IHC. Tube formation assay was also performed for examining the angiogenesis formation of the tissue in order to evaluate the possibility of a healthy organ development. RESULTS: Characterization experiments proved that the decellularized skin preserved a normal skin 3D construction and vasculature along with significant ECM arrangements. The well-kept placental ECM scaffold was cytocompatible, supportive of mesenchymal cell types. Native organ related scaffold is expected to carry a huge influence in skin tissue engineering via delivering a niche for skin-based cells and even for stem/progenitor cells. Regarding to the data obtained from this study, in vivo investigation the skin-like structure in animal models is thought to be the next step as a future prospect. CONCLUSION: This study is a reference investigation for skin engineering based on placental stem cells and biological scaffolds.

Long-Term Culture of Mouse Fetal Hepatic Stem/Progenitor Cells.

Mouse fetal liver includes abundant hepatic stem/progenitor cells (HSPCs). Easy expansion with passage of HSPCs is necessary to obtain steady data. However, it is often difficult to enrich only HSPCs, and HSPCs can die when usual trypsin is used for replating. Here, we introduce serum-free long-term culture with passage of HSPCs using fetal mouse liver without a cell sorter.

The Use of Stem Cells in Neural Regeneration: A Review of Current Opinion.

Nerve injury is a large problem that produces much pain in patients. Injury to the nervous system causes serious consequences and affects a person's quality of life. The development of tissue engineering has created a brighter future for nerve regeneration, and research has not stopped since the discovery of stem cells. Stem cells are a type of pluripotent cell that exhibits the capacity of selfdifferentiation and proliferation. Many studies have demonstrated the ability of stem cells to differentiate into other types of cells, including neurons, after induction with trophic factors in vivo and in vitro. Scientists have isolated a variety of stem cells from different organs and tissues in the human body and demonstrated that these cells were efficacious in regenerative medicine. The use of these cells provides a non-surgical method for the treatment of neurological diseases, such as nerve defects. However, many problems must be resolved before using these cells in the clinical field. The microenvironment and delivery methods of cells also affect the regeneration process. The present article comprehensively summarizes the progress of stem cells in the field of nerve regeneration in the recent decades.