Stem cells and development最新文献

Research on tooth regeneration using human-induced pluripotent stem cells (hiPSCs) is valuable for autologous dental regeneration. Acquiring mesenchymal and epithelial cells as a resource for dental regeneration is necessary because mesenchymal-epithelial interactions play an essential role in dental development. We reported the establishment of hiPSCs-derived dental epithelial-like cell (EPI-iPSCs), but hiPSCs-derived dental mesenchymal stem cells (MSCs) have not yet been reported. This study was conducted to establish hiPSCs-derived MSCs and to differentiate them into dental cells with EPI-iPSCs. Considering that dental MSCs are derived from the neural crest, hiPSCs were induced to differentiate into MSCs through neural crest formation to acquire the properties of dental MSCs. To differentiate hiPSCs into MSCs through neural crest formation, established hiPSCs were cultured and differentiated with PA6 stromal cells and differentiated hiPSCs formed neurospheres on ultralow-attachment plates. Neurospheres were differentiated into MSCs in serum-supplemented medium. Neural crest-mediated MSCs (NC-MSCs) continuously showed typical MSC morphology and expressed MSC markers. After 8 days of odontogenic induction, the expression levels of odontogenic/mineralization-related genes and dentin sialophosphoprotein (DSPP) proteins were increased in the NC-MSCs alone group in the absence of coculturing with dental epithelial cells. The NC-MSCs and EPI-iPSCs coculture groups showed high expression levels of amelogenesis/odontogenic/mineralization-related genes and DSPP proteins. Furthermore, the NC-MSCs and EPI-iPSCs coculture group yielded calcium deposits earlier than the NC-MSCs alone group. These results indicated that established NC-MSCs from hiPSCs have dental differentiation capacity with dental epithelial cells. In addition, it was confirmed that hiPSCs-derived dental stem cells could be a novel cell source for autologous dental regeneration.

Knee osteoarthritis (KOA) is a prevalent condition characterized by the progressive deterioration of the entire joint and has emerged as a prominent contributor to disability on a global scale. The nature of the disease and its impact on joint function significantly limit mobility and daily activities, highlighting its substantial influence on patients' overall well-being. Stromal vascular fraction (SVF) is a heterogenous, autologous cell product, containing mesenchymal stem cells, derived from the patient's subcutaneous adipose tissue with demonstrated safety and efficacy in the treatment of KOA patients. We conducted a single-arm, open-label, multisite, FDA approved clinical study in Kellgren-Lawrence severity grade 2-4 KOA patients. The cellular product was manufactured from patient-specific lipoaspirate in a centrally located FDA-compliant manufacturing facility. Twenty-nine subjects were treated with a quality tested single intra-articular injection of GMP manufactured SVF. Adverse events, laboratory values, vital signs, and physical examination findings were monitored during the study period. Robust tolerability, without any substantial safety issues, was demonstrated. Knee pain and function, assessed through the Knee Injury and Osteoarthritis Outcome Score (KOOS), demonstrated notable improvements. These positive benefits persisted for up to 12 months, and the majority of participants expressed satisfaction. SVF from each patient was stored in a liquid nitrogen freezer for future clinical treatments. Unique to this study of autologous cells is the shipment of lipoaspirate from the clinic to a central FDA-compliant manufacturing facility for cleanroom-controlled manufacturing. The cell product characterization data demonstrate that this method produces an equivalent product in terms of cell count and viability with the added benefit of further quality assurance testing, including sterility, endotoxin, and flow cytometry, before patient administration. Clinical Trial Registration Number: NCT04043819.

Mouse postnatal neural stem cells (pNSCs) can be expanded in vitro in the presence of epidermal growth factor and fibroblast growth factor 2 and upon removal of these factors cease proliferation and generate neurons, astrocytes, and oligodendrocytes. The genetic requirements for self-renewal and lineage-commitment of pNSCs are incompletely understood. In this study, we show that the transcription factors NFIA and NFIB, previously shown individually, to be essential for the normal commitment of pNSCs to the astrocytic lineage in vivo, are jointly required for normal self-renewal of pNSCs in vitro and in vivo. Using conditional knockout alleles of Nfia and Nfib, we show that the simultaneous loss of these two genes under self-renewal conditions in vitro reduces the expression of the proliferation markers PCNA and Ki67, eliminates clonogenicity of the cells, reduces the number of cells in S phase, and induces aberrant differentiation primarily into the neuroblast lineage. This phenotype requires the loss of both genes and is not seen upon loss of Nfia or Nfib alone, nor with combined loss of Nfia and Nfix or Nfib and Nfix. These data demonstrate a unique combined requirement for both Nfia and Nfib for pNSC self-renewal.

Seipin deficiency is an important cause of type 2 Berardinelli-Seip congenital dyslipidemia (BSCL2). BSCL2 is a severe lipodystrophy syndrome with lack of adipose tissue, hepatic steatosis, insulin resistance, and normal or higher bone mineral density. Bone marrow mesenchymal stem cells (BMSCs) are believed to maintain bone and fat homeostasis by differentiating into osteoblasts and adipocytes. We aimed to explore the role of seipin in the osteogenic/adipogenic differentiation balance of BMSCs. Seipin loxP/loxP mice are used to explore metabolic disorders caused by seipin gene mutations. Compared with wild-type mice, subcutaneous fat deficiency and ectopic fat accumulation were higher in seipin knockout mice. Microcomputed tomography of the tibia revealed the increased bone content in seipin knockout mice. We generated seipin-deficient BMSCs in vitro and revealed that lipogenic genes are downregulated and osteogenic genes are upregulated in seipin-deficient BMSCs. In addition, peroxisome proliferator-activated receptor gamma (PPARγ) signaling is reduced in seipin-deficient BMSCs, while using the PPARγ activator increased the lipogenic differentiation and decreased osteogenic differentiation of seipin-deficient BMSCs. Our findings indicated that bone and lipid metabolism can be regulated by seipin through modulating the differentiation of mesenchymal stem cells. Thus, a new insight of seipin mutations in lipid metabolism disorders was revealed, providing a prospective strategy for MSC transplantation-based treatment of BSCL2.

Over the past 15 years, there has been a significant shift in biomedical research toward a major focus on stem cell research. Although stem cells and their derivatives exhibit potential in modeling and mitigating human diseases, the ongoing objective is to enhance their utilization and translational potential. Stem cells are increasingly employed in both academic and commercial settings for a variety of in vitro and in vivo applications in regenerative medicine. Notably, accessibility to stem cell research in low-Earth orbit (LEO) has expanded, driven by the unique properties of space, such as microgravity, which cannot exactly be replicated on Earth. As private enterprises continue to grow and launch low-orbit payloads alongside government-funded spaceflight, space has evolved into a more viable destination for scientific exploration. This review underscores the potential benefits of microgravity on fundamental stem cell properties, highlighting the adaptability of cells to their environment and emphasizing physical stimuli as a key factor influencing cultured cells. Previous studies suggest that stimuli such as magnetic fields, shear stress, or gravity impact not only cell kinetics, including differentiation and proliferation, but also therapeutic effects such as cells with improved immunosuppressive capabilities or the ability to identify novel targets to refine disease treatments. With the rapid progress and sustained advocacy for space research, we propose that the advantageous properties of LEO create novel opportunities in biomanufacturing for regenerative medicine, spanning disease modeling, the development of stem cell-derived products, and biofabrication.

Solid abdominal organ transplantation is fraught with variable rates of rejection and graft versus host disease (GVHD). We sought to determine the safety and efficacy of an advanced extracellular vesicle (EV) investigational product (IP) derived from mesenchymal stem cells (MSC) in the transplant patient population. Seven separate emergency investigational new drug (eNIDs) were filed with the Food and Drug Administration (FDA) for the emergency treatment of rejection of an isolated intestinal graft (n = 2), liver allograft graft (n = 2), modified multivisceral graft (n = 3), and GVHD in isolated intestinal transplant patients (n = 2). Fifteen milliliters of IP was administered intravenously on Day 0, 2, 4, and this treatment cycle was repeated up to four times in each patient depending on the treatment protocol allowed by the FDA. Safety (adverse event reporting) and efficacy (clinical status, serologies, and histopathology) were evaluated. There were no adverse events related to IP. All patients had improvement in clinical symptoms within 24 h, improved serologic laboratory evaluation, improved pulmonary symptoms and dermatologic manifestations of GVHD, and complete histologic resolution of graft inflammation/rejection within 7 days of IP administration. Systemic use of a MSC-derived EV IP was successful in achieving histological clearance of intestinal, liver, and multivisceral graft inflammation, and skin and pulmonary manifestations of GVHD.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSC-EVs) have been proposed as a novel therapeutic tool with numerous clinically related advantages. However, their characteristics and functionality are dependent on the source of MSCs and their cell culture conditions. Fetal bovine serum (FBS) provides a source of nutrients and growth factors to the cultured cells. However, certain pitfalls are associated with its supplementation to the culture media, including introduction of exogenous FBS-derived EVs to the cultured cells. Thus, recent practices recommend utilization of serum-free (SF) media or EV-depleted FBS. On the contrary, evidence suggests that the immunomodulatory ability of MSC-EVs can be improved by exposing MSCs to an inflammatory (IF) environment. The objective of this study was to (1) compare EVs isolated from two tissue sources of MSCs that were exposed to various cell culture conditions and (2) to evaluate their anti-inflammatory effects. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) and umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were exposed to either a SF media environment, an IF environment, or media supplemented with 5% EV-depleted FBS. Following isolation of MSC-EVs, the isolates were quantified and evaluated for particle size, phenotypic changes, and their immunomodulatory potential. A statistically significant difference was not identified on the yield and protein concentration of different isolates of EVs from BM-MSCs and UC-MSCs, and all isolates had a circular appearance as evaluated via electron microscopy. A significant difference was identified on the phenotype of different EVs isolates; however, all isolates expressed classical markers such as CD9, CD63, and CD81. The addition of BM-derived MSC-EVs from FBS environment or UC-derived MSC-EVs from IF environment resulted in statistically significant downregulation of IL-6 messenger RNA (mRNA) in stimulated leukocytes. This study confirms that EVs produced by different MSC sources and cell culture conditions affect their phenotype and their immunomodulatory capacities.

Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.

Mesenchymal stem cells (MSCs) directly differentiate into neurons and endothelial cells after transplantation, and their secretome has considerable potential for treating brain injuries. Previous studies have suggested that the effects of MSCs priming with exposure to hypoxia, cytokines, growth factors, or chemical agents could optimize the paracrine potency and therapeutic potential of MSCs. Studies have suggested that thrombin-primed Wharton's Jelly-derived mesenchymal stem cells (Th.WJ-MSCs) significantly enhance the neuroprotective beneficial effects of naive MSCs in brain injury such as hypoxic-ischemic brain injury (HIE) and intraventricular hemorrhage (IVH). This study aimed to characterize WJ-MSCs in terms of stem cell markers, differentiation, cell proliferation, and paracrine factors by comparing naive and Th.WJ-MSCs. We demonstrated that compared with naive MSCs, Th.MSCs significantly enhanced the neuroprotective effects in vitro. Moreover, we identified differentially expressed proteins in the conditioned media of naive and Th.WJ-MSCs by liquid chromatography-tandem mass spectrometry analysis. Secretome analysis of the conditioned medium of WJ-MSCs revealed that such neuroprotective effects were mediated by paracrine effects with secretomes of Th.WJ-MSCs, and hepatocyte growth factor was identified as a key paracrine mediator. These results can be applied further in the preclinical and clinical development of effective and safe cell therapeutics for brain injuries such as HIE and IVH.