STEM CELLS最新文献

Optogenetics holds great potential for diverse biological applications, including fundamental research, tissue engineering, and regenerative medicine, by enabling the precise spatial and temporal control of cellular signaling pathways. Transforming growth factor-beta (TGFβ), a multifunctional cytokine, is a critical regulator of cell proliferation, differentiation, and particularly chondrogenesis. Although TGFβ signaling is necessary for effective chondrogenic differentiation, previous studies have primarily relied on recombinant TGFβ ligand supplementation. In this study, we established an advanced optogenetic platform by knocking-in opto-TGFβ receptors in the AAVS1 locus of human embryonic stem cells (hESCs), enabling precise optogenetic activation of endogenous TGFβ signaling. Blue light illumination specifically activated TGFβ signaling, indicated by enhanced SMAD2 phosphorylation. Employing a three-dimensional pellet culture system, we demonstrated that direct optogenetic activation of TGFβ receptors, without exogenous ligand supplementation, is sufficient for robust chondrogenic differentiation of hESC-derived mesenchymal stem cells. The efficiency of optogenetic differentiation was comparable to conventional recombinant TGFβ protein treatment, evidenced by the expression of chondrogenic markers and deposition of cartilage-specific extracellular matrix components, including aggrecan and type II collagen. Our findings directly confirm the sufficiency and critical role of TGFβ receptor activation itself in chondrogenesis. Furthermore, this optogenetic approach provides a theoretical advantage by enabling non-invasive external modulation of TGFβ signaling post-transplantation, potentially facilitating further maturation and functional integration of transplanted chondrocytes. Thus, our results highlight a promising recombinant-protein-free strategy for use in cartilage tissue engineering and regenerative medicine.

Stem cells use oxidized nicotinamide adenine dinucleotide (NAD+) in distinct subcellular compartments to support self-renewal and to regulate chromatin. There is limited information, however, about the biosynthetic pathways that replenish intracellular NAD+, which is continuously turned over in undifferentiated mouse embryonic stem cells. Establishing specific metabolic inputs for maintaining self-renewal can help direct reprogramming efforts. We used single fluorescent protein biosensors for in situ NAD+ measurements in J1 mouse embryonic stem cells. Sensors and controls were localized to the nucleus, cytoplasm, and mitochondrial compartments. Using a specific inhibitor for nicotinamide salvage, we found that loss of this pathway depleted NAD+ concentrations in all three subcellular compartments in undifferentiated culture conditions. We determined that loss of nicotinamide salvage reduced colony size, extended cell cycle, and resulted in diminished expression of self-renewal markers. Supplementation with precursors in the nicotinamide salvage pathway bypassed the pharmacological block, replenished cytosolic NAD+ levels, and reversed the effects on colony size. Notably, supplementation with deaminated precursors did not replenish intracellular NAD+ levels, suggesting minimal contribution from this pathway at this stage. In support, expression data from multiple mouse and human lines showed that nicotinamide salvage pathway enzyme NAMPT was predominantly expressed at the embryonic stem cell stage compared to the enzymes in other NAD+ biosynthesis pathways. Collectively, the data showed that undifferentiated embryonic stem cells heavily rely on nicotinamide salvage, indicating that this dependency is conserved.

Hematopoietic stem cell (HSC) transplantation is a potentially curative option for patients with hematologic malignancies, but donor shortages impact graft availability. Umbilical cord blood (UCB) is a viable alternative source of HSC, however the limited numbers present in a single unit have spurred efforts to expand HSC ex vivo. We previously demonstrated that the addition of valproic acid (VPA), an anti-convulsive drug, to CB cell cultures promotes maintenance of functional HSC, but not expansion. However, it has been proposed that VPA primarily induces mitochondrial reprogramming of mature CD34+CD90- cells to more primitive CD34+CD90+ cells, rather than the replication of CD34+CD90+ cells in culture. To determine which fraction of the CD34+CD90+ cells present after culture in VPA were derived from CD34+CD90- vs. CD34+CD90+ cells, we examined the functionality of CD34+CD90+ cells derived from each flow cytometry-sorted population. During culture in VPA there was a significant increase in CD34+CD90+ cell number; the majority arising from pre-existing CD34+CD90+ cells, with minimal contribution from CD34+CD90- cells. Colony-forming unit (CFU) assays revealed reduced plating efficiency and xeno-transplantation studies demonstrated diminished in vivo hematopoietic reconstitution potential of CD34+CD90+ cells derived from relatively committed CD34+CD90- cells. Our findings indicate that while VPA supports CD34+CD90+ cell expansion, the CD34+CD90+ cells derived from CD34+CD90- cells are functionally more differentiated than those derived directly from CD34+CD90+ cells, with increased mitochondrial mass and membrane potential, but reduced regenerative potential. These results emphasize the need for functional assessments of culture-expanded HSCs to accurately determine their therapeutic potential.

Osteoradionecrosis of the jaws (ORNJ) is a severe aseptic complication of high-dose radiotherapy for head-and-neck cancers, characterized by chronic jawbone necrosis, functional impairment, and poor responses to traditional treatments (e.g., hyperbaric oxygen, surgical resection) that fail to address its root pathophysiology (microcirculatory impairment, bone metabolism dysfunction). Its incidence is 1.2%-40% in patients receiving >60 Gy radiotherapy, especially with concurrent trauma. In recent years, stem cell therapy has garnered attention as a potential treatment for a variety of bone-related disorders, including the repair of bone defects, treatment of osteoarthritis, and mitigation of osteoporosis. Evidence from preclinical studies indicates that local transplantation of mesenchymal stem cells in rodent models of ORNJ significantly increases bone volume and bone mineral density. The therapeutic efficacy is primarily attributed to the cells' capacity for multidirectional differentiation, paracrine signaling, and immunomodulatory functions, highlighting their substantial potential for clinical translation. This narrative review synthesizes studies on stem cell therapy for ORNJ published from 2004 to 2024 (PubMed, Medline, Cochrane), with a focus on original research published in the most recent decade (2014-2024) to reflect the latest advances. This review consolidates ORNJ pathogenesis and stem cell mechanisms, identifies research gaps, and guides future efforts to standardize protocols and advance clinical translation.

Background: Mesenchymal stromal/stem cell (MSC) transplantation has emerged as a promising therapeutic strategy for managing cutaneous scarring, an issue associated with significant aesthetic and functional morbidity. This systematic review evaluates the potential of MSCs to modulate scarring, highlighting their efficacy and distinct mechanisms from traditional scar treatments.

Materials and methods: The review adheres to the PRISMA guidelines. We followed a prospectively registered protocol and conducted comprehensive searches in the PubMed and EMBASE databases. Eleven studies, including preclinical and clinical trials, met the inclusion criteria. Study quality was assessed using the ROBINS-I and Cochrane Risk of Bias 2 tools.

Discussion: MSC and MSC-conditioned medium therapies derived from adipose tissue, bone marrow, or the umbilical cord demonstrated significant improvements in scar appearance, reductions in thickness and volume, and beneficial remodeling of collagen structures. MSC treatment positively influenced inflammatory and immunomodulatory responses, as reflected by the regulation of cytokines and fibrotic biomarkers. However, the heterogeneity in methodologies, MSC sources, and administration routes limits the ability to make conclusive statements. Furthermore, insufficient transparency in MSC preparation challenges clinical reproducibility and application.

Conclusion: MSC therapy is becoming increasingly important in regenerative medicine. Based on our findings, MSC therapy demonstrates potential in scar remodeling through antifibrotic and immunomodulatory effects. However, robust randomized controlled trials and standardized product reporting are essential to confirm long-term efficacy and safety, improve reproducibility, and facilitate clinical translation. Advancements in these areas will define the future role of MSC therapies in managing scarring.

Introduction: The sustained production of blood and immune cells is driven by a pool of hematopoietic stem cells (HSCs) and their offspring. Due to the intrinsic heterogeneity of HSCs, the composition of emergent clones changes over time, leading to a reduced clonality in aging mice and humans. Theoretical analyses suggest that clonal conversion rates and clonal complexity depend not only on HSC heterogeneity, but also on additional stress conditions. These insights are particularly relevant in the context of stem cell transplantations, which still remain the only curative option for many hematologic diseases, increasingly considered viable for elderly individuals. However, age-related clonal changes post-transplantation are not well understood.

Methods: To address this, we conducted a barcode-based assessment of clonality to investigate post-transplantation changes in both homo- and hetero-chronic settings, combined with low- and high-intensity pre-conditioned recipients.

Results: A robust and polyclonal engraftment was observed across all groups, but with distinct differences in barcode diversity. In particular, transplanted aged HSCs showed no changes in clonality, regardless of recipient age or pre-conditioning. Young HSCs transplanted into severely pre-conditioned old hosts as well as under reduced pre-conditioning, allowed for full lymphoid reconstitution, but showed substantial differences in clonality. Also, myeloid lineage bias, a hallmark of aged HSCs, was confirmed at a clonal level across all experimental groups. Overall, we found that aged HSCs generally maintain clonal diversity similar to young HSCs, but notable differences emerge under hetero-chronic conditions and varying pre-conditioning regimens.

Conclusion: These findings challenge current paradigms and underscore the complex interactions between aging and transplantation conditions.

Importance: Mesenchymal stromal cells (MSCs) possess therapeutic properties that mediate repair. Obesity impairs MSC functionality and therapeutic efficacy, possibly by eliciting dynamic modifications of epigenetic markers, like 5-hydroxymethylcytosine (5hmC).

Objective: We hypothesized that human obesity alters the 5hmC landscape and anti-inflammatory capacity of adipose tissue-derived MSCs to activate the prominent inflammatory signaling mediator Interleukin (IL)-1β.

Design, setting, participants, intervention: Adipose tissue samples were collected from obese and lean individuals (body mass index ≥30 or <30 kg/m2, respectively, n = 11 each) during weight-loss or kidney donation surgery.

Main outcomes and measures: MSCs were harvested and analyzed for 5hmC profiles (MeDIP-seq) and mRNA expression (RNA-seq) (n = 5 each). Subsequently, MSCs or a vehicle were injected into mice, (n = 6 each) and two-weeks later, kidneys were evaluated using in-vivo magnetic resonance imaging and ex vivo studies. The role of IL-1β was then studied in-vitro in MSC-induced immunomodulation using siRNA in macrophages.

Results: Compared to MSC from lean patients, obese-MSC genes showed 2087 differential 5hmC modifications and 175 differential mRNA expression. Among them, 14 genes with overlapping alterations were involved in regulation of cytokine production, prominently IL-1β. Injecting obese MSCs elevated renal expression of IL-1β and M1 macrophage count but lowered kidney perfusion. Silencing IL-1β in obese-MSCs in vitro reduced M1 phenotype switching in co-incubated macrophages.

Conclusions and relevance: Obesity induces epigenetic and gene expression changes in MSCs, particularly in IL-1β, associated with impaired anti-inflammatory functionality of MSCs. Targeting IL-1β could be a useful therapeutic approach to modulate the decline in MSC functionality resulting from obesity.

The fate of hematopoietic stem cells (HSCs) is determined by a complex regulatory network supporting self-renewal and quiescence within a niche. Umbilical cord mesenchymal stromal cells (UC-MSCs) are classified as an alternative niche for the expansion of hematopoietic stem and progenitor cells (HSPCs). The molecular mechanisms by which UC-MSCs regulate hematopoiesis are still not fully understood. In this study, the cocultures of UC-MSCs and umbilical cord blood CD34+ (UCB-CD34+) cells were established. Immunophenotype, cell proliferation, and hematopoietic function of UCB-CD34+ cells were evaluated on days 0 to 7. UC-MSCs promoted UCB-CD34+ cell proliferation but were less effective at preserving their stemness. Notably, UC-MSCs promoted the myeloid lineage commitment, significantly observed on day 3. Integrative transcriptomic analysis highlighted the molecular signature and regulatory networks of UC-MSCs. The long non-coding RNA (lncRNA)-RNA binding protein (RBP) interaction network and lncRNA cis- and trans-regulatory networks were evident. The significant 3-gene modules and a set of 10-hub genes were identified in the protein-protein interaction (PPI) network, including RPS16, CD74, RPL35, COX7C, RPL38, RPS28, RPS27, RPS10, TARDBP, and TOMM7. These findings exemplify the niche activity of UC-MSCs in regulating cell differentiation, genomic stability maintenance, and modulation of the hematopoietic supportive niche. The transcriptional landscape, together with the identified regulatory networks, gene modules, and key hub genes provide new insights into the molecular mechanisms of UC-MSCs and establish a basis for refining ex vivo culture systems for therapeutic HSC expansion.