Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation.

BACKGROUND: Intrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA. METHODS: Human amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-ß1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection. RESULTS: qPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC. CONCLUSIONS: Our data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

IUA is the crucial cause of infertility in women of childbearing age, and no satisfactory treatment measures have been found in the clinic. hAMSCs can effectively treat intrauterine adhesions through paracrine and transdifferentiation mechanisms. This study confirmed in vitro and in vivo that amniotic mesenchymal stem cells preferentially inhibited endometrial fibrosis and promoted epithelial repair through paracrine, thus effectively treating intrauterine adhesions. The level of fibrosis marker proteins in IUA-THESCs decreased significantly after co-culturing with hAMSCs for 2 days in vitro. However, the level of epithelial marker proteins in hAMSCs increased significantly, requiring at least 6 days of co-culture. hAMSCs-CM had the same efficacy as hAMSCs in inhibiting fibrosis and promoting endometrial repair in IUA rats, supporting the idea that hAMSCs promoted endometrial remodeling through paracrine in vivo. In addition, GFP-labeled hAMSCs continuously colonized the endometrial stroma instead of the epithelium and gradually underwent apoptosis. These findings prove that hAMSCs ameliorate endometrial fibrosis of IUA via paracrine, preferentially than transdifferentiation, providing the latest insights into the precision treatment of IUA with hAMSCs and a theoretical basis for promoting the "cell-free therapy" of MSCs.

Multiomics analysis of platelet-rich plasma promoting biological performance of mesenchymal stem cells.

Mesenchymal Stem Cells are ideal seed cells for tissue repair and cell therapy and have promising applications in regenerative medicine and tissue engineering. Using Platelet-Rich Plasma as an adjuvant to create and improve the microenvironment for Mesenchymal Stem Cells growth can enhance the biological properties of Mesenchymal Stem Cells and improve the efficacy of cell therapy. However, the mechanism by which Platelet-Rich Plasma improves the biological performance of Mesenchymal Stem Cells is still unknown. In this study, by examining the effects of Platelet-Rich Plasma on the biological performance of Mesenchymal Stem Cells, combined with multiomics analysis (Transcriptomics, Proteomics and Metabolomics) and related tests, we analyzed the specific pathways, related mechanisms and metabolic pathways of Platelet-Rich Plasma to improve the biological performance of Mesenchymal Stem Cells. In an in vitro cell culture system, the biological performance of Mesenchymal Stem Cells was significantly improved after replacing Foetal Bovine Serum with Platelet-Rich Plasma, and the genes (ESM1, PDGFB, CLEC7A, CCR1 and ITGA6 et al.) related to cell proliferation, adhesion, growth, migration and signal transduction were significantly upregulated. Platelet-Rich Plasma can enhance the secretion function of MSC exosomes, significantly upregulate many proteins related to tissue repair, immune regulation and anti-infection, and enhance the repair effect of exosomes on skin injury. After replacing Foetal Bovine Serum with Platelet-Rich Plasma, Mesenchymal Stem Cells underwent metabolic reprogramming, the metabolism of amino acids and fatty acids and various signaling pathways were changed, the anabolic pathways of various proteins were enhanced. These results provide a theoretical and technical reference for optimizing the Mesenchymal Stem Cells culture system, improving the biological characteristics and clinical application effects of Mesenchymal Stem Cells.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs.

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Effect of bone-marrow-derived mesenchymal stem cells on the rate of orthodontic tooth movements in rabbits.

Mesenchymal stem cells from bone marrow, such as bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs), have been used as therapeutic alternatives to enhance remodeling in the bone. OBJECTIVE: This study aimed to evaluate the effects of BMAC and BM-MSCs on orthodontic tooth movements in rabbits. METHODS: A100- gram nickel-titanium closed-coil springs were used to initiate orthodontic tooth movement of the lower first premolars in 35 male New Zealand rabbits for 21 days. Using a split-mouth design, autologous BMAC or BM-MSCs were submucosally injected into the right sides of the lower jaw, while the left sides served as the control. On days 7, 14, and 21, a three-dimensional digital model scan was used to measure the amount of tooth movement. The microfocus computed tomography (Micro-CT) and histological findings were examined on day 0 as the baseline measurement and on days 7, 14, and 21. RESULTS: Compared to the control group, the quadrant receiving BMAC and BM-MSCs had a considerably greater amount of tooth movement. Histomorphometric analysis revealed that both BMAC and BM-MSCs had significantly higher numbers of osteoclasts and active bone-resorptive lacunae. The resorptive changes were greater in the BMAC and BM-MSCs groups than in the control group. CONCLUSION: The submucosal injection of BMAC and BM-MSCs accelerates orthodontic tooth movement (OTM) by decreasing bone density and supplying more osteoclast progenitor cells.

Recent advances in the use of extracellular vesicles from adipose-derived stem cells for regenerative medical therapeutics.

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) isolated from adipose tissue. They possess remarkable properties, including multipotency, self-renewal, and easy clinical availability. ADSCs are also capable of promoting tissue regeneration through the secretion of various cytokines, factors, and extracellular vesicles (EVs). ADSC-derived EVs (ADSC-EVs) act as intercellular signaling mediators that encapsulate a range of biomolecules. These EVs have been found to mediate the therapeutic activities of donor cells by promoting the proliferation and migration of effector cells, facilitating angiogenesis, modulating immunity, and performing other specific functions in different tissues. Compared to the donor cells themselves, ADSC-EVs offer advantages such as fewer safety concerns and more convenient transportation and storage for clinical application. As a result, these EVs have received significant attention as cell-free therapeutic agents with potential future application in regenerative medicine. In this review, we focus on recent research progress regarding regenerative medical use of ADSC-EVs across various medical conditions, including wound healing, chronic limb ischemia, angiogenesis, myocardial infarction, diabetic nephropathy, fat graft survival, bone regeneration, cartilage regeneration, tendinopathy and tendon healing, peripheral nerve regeneration, and acute lung injury, among others. We also discuss the underlying mechanisms responsible for inducing these therapeutic effects. We believe that deciphering the biological properties, therapeutic effects, and underlying mechanisms associated with ADSC-EVs will provide a foundation for developing a novel therapeutic approach in regenerative medicine.

Single high-dose intravenous injection of Wharton's jelly-derived mesenchymal stem cell exerts protective effects in a rat model of metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) is a significant epidemiological problem worldwide. It is a pre-morbid, chronic and low-grade inflammatory disorder that precedes many chronic diseases. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) could be used to treat MetS because they express high regenerative capacity, strong immunomodulatory properties and allogeneic biocompatibility. This study aims to investigate WJ-MSCs as a therapy against MetS in a rat model. METHODS: Twenty-four animals were fed with high-fat high-fructose (HFHF) diet ad libitum. After 16 weeks, the animals were randomised into treatment groups (n = 8/group) and received a single intravenous administration of vehicle, that is, 3 × 106 cells/kg or 10 × 106 cells/kg of WJ-MSCs. A healthy animal group (n = 6) fed with a normal diet received the same vehicle as the control (CTRL). All animals were periodically assessed (every 4 weeks) for physical measurements, serum biochemistry, glucose tolerance test, cardiovascular function test and whole-body composition. Post-euthanasia, organs were weighed and processed for histopathology. Serum was collected for C-reactive protein and inflammatory cytokine assay. RESULTS: The results between HFHF-treated groups and healthy or HFHF-CTRL did not achieve statistical significance (α = 0.05). The effects of WJ-MSCs were masked by the manifestation of different disease subclusters and continuous supplementation of HFHF diet. Based on secondary analysis, WJ-MSCs had major implications in improving cardiopulmonary morbidities. The lungs, liver and heart show significantly better histopathology in the WJ-MSC-treated groups than in the untreated CTRL group. The cells produced a dose-dependent effect (high dose lasted until week 8) in preventing further metabolic decay in MetS animals. CONCLUSIONS: The establishment of safety and therapeutic proof-of-concept encourages further studies by improving the current therapeutic model.

Harnessing three-dimensional porous chitosan microsphere embedded with adipose-derived stem cells to promote nerve regeneration.

BACKGROUND: Nerve guide conduits are a promising strategy for reconstructing peripheral nerve defects. Improving the survival rate of seed cells in nerve conduits is still a challenge and microcarriers are an excellent three-dimensional (3D) culture scaffold. Here, we investigate the effect of the 3D culture of microcarriers on the biological characteristics of adipose mesenchymal stem cells (ADSCs) and to evaluate the efficacy of chitosan nerve conduits filled with microcarriers loaded with ADSCs in repairing nerve defects. METHODS: In vitro, we prepared porous chitosan microspheres by a modified emulsion cross-linking method for loading ADSCs and evaluated the growth status and function of ADSCs. In vivo, ADSCs-loaded microcarriers were injected into chitosan nerve conduits to repair a 12 mm sciatic nerve defect in rats. RESULTS: Compared to the conventional two-dimensional (2D) culture, the prepared microcarriers were more conducive to the proliferation, migration, and secretion of trophic factors of ADSCs. In addition, gait analysis, neuro-electrophysiology, and histological evaluation of nerves and muscles showed that the ADSC microcarrier-loaded nerve conduits were more effective in improving nerve regeneration. CONCLUSIONS: The ADSCs-loaded chitosan porous microcarrier prepared in this study has a high cell engraftment rate and good potential for peripheral nerve repair.

Non-Clinical Safety Evaluation of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells in Cynomolgus Monkeys.

Purpose: In recent years, exosomes have been proved to be used to treat many diseases. However, due to the lack of uniform quality control standards for exosomes, the safety of exosomes is still a problem to be solved, especially now more and more exosomes are used in clinical trials, and its non-clinical safety evaluation is particularly important. However, there is no safety evaluation standard for exosomes at present. Therefore, this study will refer to the evaluation criteria of therapeutic biological products, adopt non-human primates to evaluate the non-clinical safety of human umbilical cord mesenchymal stem cell exosomes from the general pharmacology and immunotoxicity, aiming at establishing a safety evaluation system of exosomes and providing reference for the clinical application of exosomes in the future. Methods: 3.85 × 1012 exosomes derived from human umbilical cord mesenchymal stem cells were injected into cynomolgus monkeys intravenously. The changes of general clinical conditions, hematology, immunoglobulin, Th1/Th2 cytokines, T lymphocytes and B lymphocytes, and immune organs were observed before and within 14 days after injection. Results: The results showed that exosomes did not have obvious pathological effects on the general clinical conditions, blood, coagulation function, organ coefficient, immunoglobulin, Th1/Th2 cytokines, lymphocytes, major organs, and major immune organs (spleen, thymus, bone marrow) of cynomolgus monkeys. However, the number of granulocyte-macrophage colonies in exosomes group was significantly higher than that in control group. Conclusion: To sum up, the general pharmacological results and immunotoxicity results showed that the injection of 3.85 × 1012 exosomes may have no obvious adverse reactions to cynomolgus monkeys. This dose of exosomes is relatively safe for treatment, which provides basis research for non-clinical safety evaluation of exosomes and provides reliable research basis for future clinical application of exosomes.

Targeted silencing of GNAS in a human model of osteoprogenitor cells results in the deregulation of the osteogenic differentiation program.

Introduction: The dysregulation of cell fate toward osteoprecursor cells associated with most GNAS-based disorders may lead to episodic de novo extraskeletal or ectopic bone formation in subcutaneous tissues. The bony lesion distribution suggests the involvement of abnormal differentiation of mesenchymal stem cells (MSCs) and/or more committed precursor cells. Data from transgenic mice support the concept that GNAS is a crucial factor in regulating lineage switching between osteoblasts (OBs) and adipocyte fates. The mosaic nature of heterotopic bone lesions suggests that GNAS genetic defects provide a sensitized background for ectopic osteodifferentiation, but the underlying molecular mechanism remains largely unknown. Methods: The effect of GNAS silencing in the presence and/or absence of osteoblastic stimuli was evaluated in the human L88/5 MSC line during osteodifferentiation. A comparison of the data obtained with data coming from a bony lesion from a GNAS-mutated patient was also provided. Results: Our study adds some dowels to the current fragmented notions about the role of GNAS during osteoblastic differentiation, such as the premature transition of immature OBs into osteocytes and the characterization of the differences in the deposed bone matrix. Conclusion: We demonstrated that our cell model partially replicates the in vivo behavior results, resulting in an applicable human model to elucidate the pathophysiology of ectopic bone formation in GNAS-based disorders.

Instantly adhesive and ultra-elastic patches for dynamic organ and wound repair.

Bioadhesive materials and patches are promising alternatives to surgical sutures and staples. However, many existing bioadhesives do not meet the functional requirements of current surgical procedures and interventions. Here, we present a translational patch material that exhibits instant adhesion to tissues (2.5-fold stronger than Tisseel, an FDA-approved fibrin glue), ultra-stretchability (stretching to >300% its original length without losing elasticity), compatibility with rapid photo-projection (<2 min fabrication time/patch), and ability to deliver therapeutics. Using our established procedures for the in silico design and optimization of anisotropic-auxetic patches, we created next-generation patches for instant attachment to tissues while conforming to a broad range of organ mechanics ex vivo and in vivo. Patches coated with extracellular vesicles derived from mesenchymal stem cells demonstrate robust wound healing capability in vivo without inducing a foreign body response and without the need for patch removal that can cause pain and bleeding. We further demonstrate a single material-based, void-filling auxetic patch designed for the treatment of lung puncture wounds.

Enhanced osteochondral repair by leukocyte-depleted platelet-rich plasma in combination with adipose-derived mesenchymal stromal cells encapsulated in a three-dimensional photocrosslinked injectable hydrogel in a rabbit model.

INTRODUCTION: Intra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model. METHODS: Thirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume. RESULTS: In terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect. CONCLUSION: Regeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration.

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.

MALAT1/miR-7-5p/TCF4 Axis Regulating Menstrual Blood Mesenchymal Stem Cells Improve Thin Endometrium Fertility by the Wnt Signaling Pathway.

Thin endometrium (TE) is a significant factor contributing to fertility challenges, and addressing this condition remains a central challenge in reproductive medicine. Menstrual blood-derived mesenchymal stem cells (MenSCs) play a crucial role in tissue repair and regeneration, including that of TE. The Wnt signaling pathway, which is highly conserved and prevalent in eukaryotes, is essential for cell proliferation, tissue development, and reproductive functions. MALAT1 is implicated in various transcriptional and molecular functions, including cell proliferation and metastasis. However, the combined effects of the Wnt signaling pathway and the long non-coding RNA (lncRNA) MALAT1 on the regulation of MenSCs' regenerative capabilities in tissue engineering have not yet been explored. To elucidate the regulatory mechanism of MALAT1 in TE, we analyzed its expression levels in normal endometrium and TE tissues, finding that low expression of MALAT1 was associated with poor clinical prognosis. In addition, we conducted both in vitro and in vivo functional assays to examine the role of the MALAT1/miR-7-5p/TCF4 axis in cell proliferation and migration. Techniques such as dual-luciferase reporter assay, fluorescent in situ hybridization, and immunoblot experiments were utilized to clarify the molecular mechanism. To corroborate these findings, we established a TE model and conducted pregnancy experiments, demonstrating a strong association between MALAT1 expression and endometrial fertility. In conclusion, our comprehensive study provides strong evidence supporting that lncRNA MALAT1 modulates TCF4 expression in the Wnt signaling pathway through interaction with miR-7-5p, thus enhancing MenSCs-mediated improvement of TE and improving fertility.

Mesenchymal Stem Cell Exosomes Enhance Posterolateral Spinal Fusion in a Rat Model.

Spinal fusion, a common surgery performed for degenerative lumbar conditions, often uses recombinant human bone morphogenetic protein 2 (rhBMP-2) that is associated with adverse effects. Mesenchymal stromal/stem cells (MSCs) and their extracellular vesicles (EVs), particularly exosomes, have demonstrated efficacy in bone and cartilage repair. However, the efficacy of MSC exosomes in spinal fusion remains to be ascertained. This study investigates the fusion efficacy of MSC exosomes delivered via an absorbable collagen sponge packed in a poly Ɛ-caprolactone tricalcium phosphate (PCL-TCP) scaffold in a rat posterolateral spinal fusion model. Herein, it is shown that a single implantation of exosome-supplemented collagen sponge packed in PCL-TCP scaffold enhanced spinal fusion and improved mechanical stability by inducing bone formation and bridging between the transverse processes, as evidenced by significant improvements in fusion score and rate, bone structural parameters, histology, stiffness, and range of motion. This study demonstrates for the first time that MSC exosomes promote bone formation to enhance spinal fusion and mechanical stability in a rat model, supporting its translational potential for application in spinal fusion.

Mesenchymal Stem Cell Therapy for Bone Repair of Human Hip Osteonecrosis with Bilateral Match-Control Evaluation: Impact of Tissue Source, Cell Count, Disease Stage, and Volume Size on 908 Hips.

We investigated the impact of mesenchymal stem cell (MSC) therapy on treating bilateral human hip osteonecrosis, analyzing 908 cases. This study assesses factors such as tissue source and cell count, comparing core decompression with various cell therapies. This research emphasizes bone repair according to pre-treatment conditions and the specificities of cell therapy in osteonecrosis repair, indicating a potential for improved bone repair strategies in hips without femoral head collapse. This study utilized a single-center retrospective analysis to investigate the efficacy of cellular approaches in the bone repair of osteonecrosis. It examined the impact on bone repair of tissue source (autologous bone marrow concentrate, allogeneic expanded, autologous expanded), cell quantity (from none in core decompression alone to millions in cell therapy), and osteonecrosis stage and volume. Excluding hips with femoral head collapse, it focused on patients who had bilateral hip osteonecrosis, both pre-operative and post-operative MRIs, and a follow-up of over five years. The analysis divided these patients into seven groups based on match control treatment variations in bilateral hip osteonecrosis, primarily investigating the outcomes between core decompression, washing effect, and different tissue sources of MSCs. Younger patients (<30 years) demonstrated significantly better repair volumes, particularly in stage II lesions, than older counterparts. Additionally, bone repair volume increased with the number of implanted MSCs up to 1,000,000, beyond which no additional benefits were observed. No significant difference was observed in repair outcomes between different sources of MSCs (BMAC, allogenic, or expanded cells). The study also highlighted that a 'washing effect' was beneficial, particularly for larger-volume osteonecrosis when combined with core decompression. Partial bone repair was the more frequent event observed, while total bone repair of osteonecrosis was rare. The volume and stage of osteonecrosis, alongside the number of injected cells, significantly affected treatment outcomes. In summary, this study provides comprehensive insights into the effectiveness and variables influencing the use of mesenchymal stem cells in treating human hip osteonecrosis. It emphasizes the potential of cell therapy while acknowledging the complexity and variability of results based on factors such as age, cell count, and disease stage.

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.

Osteoinductive micro-nano guided bone regeneration membrane for in situ bone defect repair.

BACKGROUND: Biomaterials used in bone tissue engineering must fulfill the requirements of osteoconduction, osteoinduction, and osseointegration. However, biomaterials with good osteoconductive properties face several challenges, including inadequate vascularization, limited osteoinduction and barrier ability, as well as the potential to trigger immune and inflammatory responses. Therefore, there is an urgent need to develop guided bone regeneration membranes as a crucial component of tissue engineering strategies for repairing bone defects. METHODS: The mZIF-8/PLA membrane was prepared using electrospinning technology and simulated body fluid external mineralization method. Its ability to induce biomimetic mineralization was evaluated through TEM, EDS, XRD, FT-IR, zeta potential, and wettability techniques. The biocompatibility, osteoinduction properties, and osteo-immunomodulatory effects of the mZIF-8/PLA membrane were comprehensively evaluated by examining cell behaviors of surface-seeded BMSCs and macrophages, as well as the regulation of cellular genes and protein levels using PCR and WB. In vivo, the mZIF-8/PLA membrane's potential to promote bone regeneration and angiogenesis was assessed through Micro-CT and immunohistochemical staining. RESULTS: The mineralized deposition enhances hydrophilicity and cell compatibility of mZIF-8/PLA membrane. mZIF-8/PLA membrane promotes up-regulation of osteogenesis and angiogenesis related factors in BMSCs. Moreover, it induces the polarization of macrophages towards the M2 phenotype and modulates the local immune microenvironment. After 4-weeks of implantation, the mZIF-8/PLA membrane successfully bridges critical bone defects and almost completely repairs the defect area after 12-weeks, while significantly improving the strength and vascularization of new bone. CONCLUSIONS: The mZIF-8/PLA membrane with dual osteoconductive and immunomodulatory abilities could pave new research paths for bone tissue engineering.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

A GMP-compliant manufacturing method for Wharton's jelly-derived mesenchymal stromal cells.

BACKGROUND: Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. METHODS: Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). RESULTS: The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20-27 °C. CONCLUSION: This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.