Stem Cells International最新文献

Recently, we used kinetic stem cell (KSC) counting to show that the stability of the stem cell fraction (SCF) during serial culture expansion of human oral alveolar bone mesenchymal tissue cell (MTC) preparations varied significantly among patient donors. Whereas some patient donor samples' SCFs declined rapidly, others showed a moderate decrease; and still others had highly stable SCFs during serial culture expansion. Defining the cell kinetics basis for these differences in SCF stability could lead to effective solutions for the problem of loss of mesenchymal stem cell (MSC) function when MTC preparations are expanded for research and clinical applications. Using KSC counting, we show that greater SCF stability is associated with smaller stem cell turnover units, which reduce the SCF by cell dilution. This finding confirms earlier evidence and proposals that, by limiting the production and proliferation of committed progenitor cells, more effective expansion of tissue stem cells, such as MSCs, can be achieved. Because of the universal nature of tissue stem cell turnover units and the continuation of their basic cell kinetics programs in in vitro cell culture, the effects defined herein are predicted to apply to other types of human tissue stem cells of interest for stem cell medicine.

Recurrent pregnancy loss (RPL), defined as more than two consecutive miscarriages before 20 weeks of gestation, affects 1%-5% of reproductive-aged women, with nearly half of the cases remaining idiopathic. Using ethanol-induced endometrial injury in rats to simulate RPL, we demonstrated that mesenchymal stem cells (MSCs) exert therapeutic effects through two complementary mechanisms: CCR7-mediated endometrial repair and immunomodulation of uterine natural killer (uNK) cells, compared to ethanol-induced injury, this treatment achieved a 190% increase in embryo retention (from 2.4 to 7 embryos). Transcriptomic analysis and Western blotting of MSC-cocultured NK92 cells revealed significant CCR7 upregulation (1.75-fold increase at the optimal dose of 2 × 10⁴ cells/mL MSCs) and activation of NK differentiation pathways. This was corroborated by immunofluorescence showing enhanced CCR7⁺ NK cell infiltration in MSC-treated endometria. MSCs administration altered cytokine profiles by decreasing pro-inflammatory mediators (IL-6, TNF-α, and IL-1β) and increasing anti-inflammatory IL-10 levels simultaneously. Mechanistically, MSCs orchestrate endometrial repair through sequential events: induce the formation of a gradient of CCR7 expression on the endometrial layer and the surface of NK cells; followed by ERK/JNK pathway activation, which promotes CCR7⁺ uNK cell generation; and finally initiates endometrial proliferation with an increased proportion of Ki67⁺ cells. Our integrated multi-omics approach-combining RNA-Seq, protein analysis, and cytokine profiling-establishes the CCR7-ERK/JNK axis as a promising therapeutic target, providing clinically relevant parameters for MSCs dosing and administration protocols in idiopathic RPL management.

Erectile dysfunction (ED) is widespread among individuals with high blood pressure and negatively affects quality of life. The effect of stromal vascular fraction (SVF) on hypertension-related ED remains unexplored. We used a hypertensive rat model to explore the relative efficacy of adipose tissue stromal vascular fraction (tSVF) and cellular SVF (cSVF). We then investigated the possible mechanisms of these treatments. Hypertensive rats were divided into three groups according to different treatments. Their intracavernous pressure (ICP) during erection and condition of cavernous tissue were compared to those of the controlled group. Endothelial-mesenchymal transformation (EndMT) markers as well as related inflammatory factors were also measured. cSVF and tSVF were labeled with CM-Dil before injection in order to determine whether cSVF and tSVF survived, proliferated, and transdifferentiated in vivo. The increased ICP during erection demonstrated that tSVF treatment significantly improved hypertension-related ED. tSVF increased the smooth muscle-to-collagen ratio and inhibiting the expression of fibrosis-related proteins in hypertensive rats while rescuing the expression of vWF and eNOS, which indicated the preserving of endothelial tissue of the penis. Immunofluorescence staining and western blotting of penile tissue clearly suggest the inhibitory effect of tSVF on the overoccurring EndMT. Immunofluorescence staining and Western blot analysis of endothelial cells in vitro corroborate the whole-tissue findings. The experiments in N-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced human umbilical vein endothelial cells (HUVECs) revealed tSVF suppresses EndMT via inhibiting the TGF-β2-Smad2/Smad3 pathway. In vivo tSVF and cSVF tracing suggested that tSVF showed better longevity and transdifferentiation capacity than cSVF, thus exerting a more significant therapeutic effect. Treatment with tSVF significantly reserved erectile function in a hypertensive rat model. The mechanism appears to be inhibition of pathological EndMT through self-differentiation. We conclude that tSVF is a promising therapeutic candidate for treating hypertensive ED.

Background: Bronchopulmonary dysplasia (BPD) remains a significant challenge in the management of preterm infants. Although conventional treatment approaches have helped reduce the morbidity associated with BPD, the prevalence of the condition has not decreased, highlighting an urgent need for new therapies. Mesenchymal stromal cell (MSC) therapy has emerged as a potentially promising intervention, showing a favorable safety profile in Phase II clinical trials. However, research on the use of MSCs as a late-term therapeutic strategy for established BPD is still in its early stages, indicating a need for further studies to evaluate their effectiveness and optimal application.

Aim: To investigate the results of seven extremely preterm infants who underwent an MSC therapy for severe established BPD.

Method: A cohort of seven infants diagnosed with severe BPD (sBPD) received MSC therapy to assist in their transition to spontaneous breathing. For the first six infants, MSC therapy was discontinued after extubation; however, the final infant continued to receive MSC therapy as he remained on nasal continuous positive airway pressure (nCPAP).Each treatment cycle involved administering 1 million MSCs per kilogram via intratracheal injection, along with an additional 0.5 million MSCs delivered through intravenous infusion. Treatment was initiated between postnatal days 32 and 84. The first three infants each underwent two treatment cycles, the fourth infant received three cycles, and the last three infants were scheduled for 4 weekly cycles. A retrospective analysis was conducted to evaluate the outcomes of the therapy.

Results: All infants were successfully extubated to nCPAP following MSC treatment. However, two infants who underwent two cycles of MSC therapy could not be weaned off respiratory support. In contrast, all infants who received three to four cycles were successfully weaned off the ventilators and discharged home without the need for supplemental oxygen. Additionally, secondary benefits were observed, including improvements in intraventricular hemorrhage (IVH) and retinopathy of prematurity (ROP), a decrease in the number of packed red blood cell transfusions, and fewer episodes of sepsis.

Conclusions: Our findings indicate that administering up to four treatment cycles may be more effective for the long-term management of sBPD. Additionally, using MSCs through both intratracheal and intravenous routes could offer benefits beyond just the lungs, highlighting an area for further research.

Background: Adipose-derived mesenchymal stem cells (ADSCs) have great potential in the realm of tissue repair and regenerative medicine. However, the exact effects of ADSCs on the healing of skin wounds and the underlying mechanisms remain unexplored. Here, we investigated the effects of ADSCs on fibroblasts and keratinocytes and their related molecular mechanisms in wound healing.

Methods: We used a murine model in vivo and a Transwell coculture system in vitro. The proliferation and migration abilities of human dermal fibroblasts (HDFs) and human immortalized keratinocytes (HaCaT) were analyzed after coculture with ADSCs, and the target molecules were investigated by transcriptome sequencing. We further investigated phenotypic changes by knocking down and overexpressing the target molecule and analyzed the potential mechanisms.

Results: We successfully extracted, expanded, and identified ADSCs. ADSCs not only accelerated wound healing in mice but also improved healing quality. Coculture with ADSCs augmented the proliferation and migration capacities of main skin cells in vitro. RNA sequencing analysis revealed that the level of serpin family E member 1 (SERPINE1) in both HDF and HaCaT was significantly regulated by ADSCs. Knockdown of SERPINE1 restrained the proliferation and migration phenotypes of HDF and HaCaT, while overexpression of SERPINE1 did exactly the opposite. Pathway enrichment analysis revealed that SERPINE1 was mainly related to PI3K-Akt and MAPK signaling pathways.

Conclusion: The in vivo model and in vitro cell test demonstrate that ADSC effectively promotes cutaneous wound healing by augmenting the proliferation and migration of fibroblasts and keratinocytes through upregulating SERPINE1, which provides novel insights into the biological roles of SERPINE1 in wound healing and suggests ADSC has a promising future in skin injury therapy.

Limbal stem cell deficiency (LSCD) results from the loss or dysfunction of limbal stem cells, posing a significant challenge due to limited treatment options. Autologous oral mucosal epithelial cell (OMEC) sheet transplantation is an innovative therapy, but its effectiveness in smokers remains unclear. This study aims to investigate the impact of smoking on the efficacy of autologous OMEC sheet transplantation for LSCD and explores how acrolein, a major cigarette smoke component, affects the biological properties of these cells. This retrospective cohort study included 13 LSCD patients (13 eyes), divided into never smokers (seven eyes) and smokers (six eyes), all of whom received autologous OMEC sheet transplantation. The study compared colony-forming abilities and sheet thickness between the groups, assessed corneal epithelial repair postoperatively, and conducted in vitro experiments treating human OMECs (hOMECs) with acrolein. Evaluations focused on colony-forming ability, stem cell marker protein P63 expression, and the secretion of repair factors (transforming growth factor-beta [TGF-β], basic fibroblast growth factor [bFGF], and hepatocyte growth factor [HGF]) as well as the wound healing potential in human corneal epithelial cells. Postoperative results showed significant improvements in corneal epithelial defects, neovascularization, and best visual acuity in never smokers. However, adhesions recurred in both groups, with earlier recurrence in smokers. In vitro, acrolein significantly inhibited the proliferation of OMECs, reduced P63 expression, and decreased the secretion of TGF-β and HGF, though bFGF levels remained unchanged, impairing wound healing in scratched corneal epithelial cells. Smoking adversely affects the efficacy of autologous OMEC transplantation for LSCD, with acrolein as a potential key factor. Future research should focus on understanding the mechanisms by which smoking impacts OMECs and developing improved therapeutic strategies for smokers with LSCD. Trial Registration: ClinicalTrials.gov identifier: NCT03015779.

Background: Severe osteonecrosis of the femoral head (ONFH) secondary to corticosteroid therapy in symptomatic, hematological young patients currently has no therapeutic alternative, and early total hip replacement (THR) is a high-risk intervention in those patients.

Objectives: To evaluate feasibility, safety, and early efficacy of allogeneic expanded mesenchymal stromal cells (MSCs) in a pilot clinical trial.

Methods: Pilot phase 1 open, noncontrolled, nonrandomized clinical trial evaluating the bone regeneration capacity in seven hips from four patients (young females 11-19 year old) with symptomatic, severe bilateral femoral head osteonecrosis (secondary to corticosteroid therapy), 1 year after being surgically treated with 140 × 10⁶ allogenic MSC plus forage.

Results: The proposed therapy proved feasibility, safety at 1 and 4 years (no related serious adverse events [SAEs]), and early efficacy (nonsignificant) in the case analysis (5/7 hips avoiding THR at 4 years).

Conclusions: The implantation of expanded allogeneic MSC in young patients to prevent conversion to a THR or collapse of the femoral head due to severe osteonecrosis is feasible without safety concerns in the longer-term follow-up (FU) upto 4 years. Trial Registration: EudraCT number: 2018-000886-35.

Background: Mesenchymal stromal cells (MSCs) support tissue repair in osteoarthritis (OA), with migration to damaged tissue being a key strategy in in situ tissue engineering. Their regenerative potential depends on factors such as differentiation, level of senescence, and responsiveness to signaling molecules. However, previous findings on these properties of OA MSCs remain inconclusive. This study integrates multiple aspects and tests feasibility using a well-characterized chemoattractant.

Methods and results: MSCs from non-OA donor (ND) and OA donor were characterized for their trilineage differentiation potential as well as for their senescence level by (immune-) histochemistry, RT-qPCR, microarray analysis, and a bead-based immunoassay for cell culture supernatants. No difference in differentiation and senescence level was observed, the latter being indicated by a similar activity of β-Galactosidase (β-gal), gene expression profiles of cyclin-dependent kinase (CDKN) inhibitor 2A (CDKN2A), CDKN inhibitor 1A (CDKN1A), sirtuin 1 (SIRT1), and matrix metallopeptidase 1 (MMP1), as well as secreted cytokines. Chemokine receptors in OA MSCs were detected using immunohistochemistry and RT-qPCR. Expression of CCR1-CCR7, CCR9, and CXCR1-CXCR6 in OA MSCs was confirmed on gene and protein levels. Both OA and ND MSCs migrated toward 1000 nM CCL25, as evaluated via a Boyden chamber assay. Subsequent genome-wide microarray analysis of OA MSCs after treatment with 1000 nM CCL25 corroborated its influence on migration, proliferation, apoptosis, and differentiation as defined by Gene Ontology terms (GO terms). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis confirmed this broad impact and emphasized the role of cytokine-cytokine-receptor interaction and metabolic pathways.

Conclusion: Our data indicate that OA MSCs retain their differentiation potential and do not exhibit an increased senescent phenotype. Their chemokine receptor profile is conducive of migration, and both OA and ND MSCs respond to CCL25, highlighting the potential of OA MSCs for directed in situ repair.

Human dental pulp stem cells (hDPSCs) hold significant promise for bone regeneration, yet efficient osteogenic induction remains challenging. Phosphorylated oligosaccharides of calcium (POs-Ca), a novel calcium salt derived from potato starch, has recently attracted attention for its remineralization capabilities and potential to promote stem cell differentiation. Here, we investigated the impact of POs-Ca on the osteogenic differentiation of hDPSCs and its underlying mechanism. Isolated hDPSCs were characterized via flow cytometry based on mesenchymal surface markers. Biocompatibility and osteogenic differentiation were assessed via Cell Counting Kit-8 (CCK-8) assay, alkaline phosphatase (ALP) activity, Alizarin Red S staining, and protein levels of osteogenic (Collagen I, DSPP, DMP1, and RUNX2). Intracellular Ca²⁺ flux was monitored using Fluo-4 AM, while AMP-activated protein kinase (AMPK) signaling and autophagic flux were analyzed by western blot (p-AMPK, p-ULK1, and light chain 3 (LC3)-II), TEM, and LC3-GFP imaging. Mechanistic studies employed verapamil (Ca²⁺ channel blocker), Compound C (CC;AMPK inhibitor), and chloroquine (CQ;autophagy inhibitor). POs-Ca (5 mg/mL) exhibited excellent biocompatibility and significantly promoted osteogenic differentiation, as evidenced by a 3.22-fold increase in ALP activity and markedly enhanced mineralized nodule formation as shown by Alizarin Red S staining. Mechanistic studies revealed that POs-Ca triggers rapid intracellular Ca²⁺ influx, activating the AMPK pathway and inducing autophagic flux. Pharmacological inhibition established the essential causality of this cascade: verapamil abolished osteogenic enhancement, while CC and CQ suppressed ALP activity, mineralization, and osteogenic marker expression. Notably, CQ reciprocally attenuated POs-Ca-induced Ca²⁺ influx, revealing novel bidirectional Ca²⁺-autophagy crosstalk. In conclusion, POs-Ca might promote hDPSCs osteogenesis via a calcium influx-driven AMPK-autophagy axis, providing a foundation for novel biomaterials that exploit physiological calcium signaling. These findings offer immediate translational potential for developing minimally invasive, cost-effective strategies in dental pulp regeneration and bone defect repair.

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) ameliorate motor deficits in cerebral palsy (CP), but the effect of injection frequency remains unclear. Moreover, most studies have focused on mild CP models (unilateral carotid artery occlusion [UCAO] model). This study explored the effect and mechanism of hUC-MSCs in a rat model of moderate-to-severe CP (bilateral carotid artery occlusion [BCAO] model). On postnatal Day 4 (P4), Wistar rat pups underwent BCAO induction. Subsequently, they received either a single intrathecal injection of hUC-MSCs on P21 or repeated injections on P21, P28, P35, and P42. Motor performance was assessed using the rotarod and front-limb suspension tests, while neuronal regeneration and inflammation were evaluated via biomarkers including neuronal nuclear antigen (NeuN), ionized calcium-binding adapter molecule-1 (Iba-1), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and brain-derived neurotrophic factor (BDNF). P18 model screening confirmed that the BCAO model resulted in more severe brain damage and motor impairment than the UCAO model. After injection of lentivirally transfected hUC-MSCs, it was found that hUC-MSCs could nest in the damaged area and survive for at least 3 days. Administration of hUC-MSCs following BCAO modeling led to notable improvements in both behavioral performance and histological outcomes. Furthermore, repeated injections offered greater therapeutic benefits compared to single injection. It indicated that the efficacy of repeated injections of hUC-MSCs in the treatment of moderate-to-severe CP was superior to that of single injection. Its mechanism was related to the improvement of damaged myelin structure, reduced immunoinflammatory responses, and increased neurotrophic support.