Human Placenta-derived Mesenchymal Stem Cells Research Articles

Human placenta-derived mesenchymal stem cells transplantation in patients with acute respiratory distress syndrome (ARDS) caused by COVID-19 (phase I clinical trial): safety profile assessment

BackgroundHigh morbidity and mortality rates of the COVID-19 pandemic have made it a global health priority. Acute respiratory distress syndrome (ARDS) is one of the most important causes of death in COVID-19 patients. Mesenchymal stem cells have been the subject of many clinical trials for the treatment of ARDS because of their immunomodulatory, anti-inflammatory, and regenerative potentials. The aim of this phase I clinical trial was the safety assessment of allogeneic placenta-derived mesenchymal stem cells (PL-MSCs) intravenous injection in patients with ARDS induced by COVID-19.MethodsWe enrolled 20 patients suffering from ARDS caused by COVID-19 who had been admitted to the intensive care unit. PL-MSCs were isolated and propagated using a xeno-free/GMP compliant protocol. Each patient in the treatment group (N = 10) received standard treatment and a single dose of 1 × 106 cells/kg PL-MSCs intravenously. The control groups (N = 10) only received the standard treatment. Clinical signs and laboratory tests were evaluated in all participants at the baseline and during 28 days follow-ups.ResultsNo adverse events were observed in the PL-MSC group. Mean length of hospitalization, serum oxygen saturation, and other clinical and laboratory parameters were not significantly different in the two groups (p > 0.05).ConclusionOur results demonstrated that intravenous administration of PL-MSCs in patients with COVID-19 related ARDS is safe and feasible. Further studies whit higher cell doses and repeated injections are needed to evaluate the efficacy of this treatment modality.Trial registration: Iranian Registry of Clinical Trials (IRCT); IRCT20200621047859N4. Registered 1 March 2021, https://en.irct.ir/trial/52947.

Human Placental Mesenchymal Stem Cells for the Treatment of ARDS in Rat.

The acute respiratory distress syndrome (ARDS) is one of the main causes of high mortality in patients with coronavirus (COVID-19). In recent years, due to the coronavirus pandemic, the number of patients with ARDS has increased significantly. Unfortunately, until now, there are no effective treatments for ARDS caused by COVID-19. Many drugs are either ineffective or have a low effect. Currently, there have been reports of efficient use of mesenchymal stem cells (MSCs) for the treatment of ARDS caused by COVID-19. We investigated the influence of freeze-dried human placenta-derived mesenchymal stem cells (HPMSCs) in ARDS rat model. All animals have received intratracheal injection of 6 mg/kg of lipopolysaccharide (LPS). The rats were randomly divided into five groups: I: LPS, II: LPS+dexamethasone, III: LPS+HPMSCs, IV: HPMSC, and V: saline. ARDS observation time was short-term and amounted to 168 hours. The study has shown that HPMSCs are able to migrate and attach to damaged lung tissue, contributing to the resolution of pathology, restoration of function, and tissue repair in the alveolar space. Studies have also shown that the administration of HPMSCs in animals with ARDS model significantly reduced the levels of key cytokines such as IL-1β, IL-6, and TNF-α. Freeze-dried placental stem cell is a very promising biomaterial for the treatment of ARDS. The human placenta can be easily obtained because it is considered as a medical waste. At the same time, a huge number of MSCs can be obtained from the placental tissue, and there is no ethical controversy around their use. The freeze-dried MSCs from human placental tissue can be stored sterile at room temperature for a long time before use.

Emerging Role of Mitochondrial Function Enhancement in Mesenchymal Stem Cell Therapy

The impact of inflammatory environments on the impaired features of MSCs, such as loss of homeostasis and stem cell senescence, still remains unclear. In the paper titled “MIT-001 Restores Human Placenta-Derived Mesenchymal Stem Cells by Enhancing Mitochondrial Quiescence and Cytoskeletal Organization”, Yu et al., investigated the phenotypes of MSCs exposed to pro-inflammatory cytokines TNF-α and IFN-γ. They further tested whether the exposed MSCs are protected from senescence by MIT-001, a novel necrosis inhibitor and an anti-inflammatory molecule

Differential Expression Profiling of microRNAs in Human Placenta-Derived Mesenchymal Stem Cells Cocultured with Grooved Porous Hydroxyapatite Scaffolds.

Scaffold materials used for bone defect repair are often limited by osteogenic efficacy. Moreover, microRNAs (miRNAs) are involved in regulating the expression of osteogenic-related genes. In previous studies, we verified the enhancement of osteogenesis using a grooved porous hydroxyapatite scaffold (HAG). In the present study, we analyzed the contribution of HAG to the osteogenic differentiation of human placenta-derived mesenchymal stem cells (hPMSCs) from the perspective of miRNA differential expression. Furthermore, results showed that miRNAs were differentially expressed in the osteogenic differentiation of hPMSCs cocultured with HAG. In detail, 16 miRNAs were significantly upregulated and 29 miRNAs were downregulated with HAG. In addition, bioinformatics analyses showed that the differentially expressed miRNAs were enriched in a variety of biological processes, including signal transduction, cell metabolism, cell junctions, cell development and differentiation, and that they were associated with osteogenic differentiation through axon guidance, mitogen-activated protein kinase, and the transforming growth factor beta signaling pathway. Furthermore, multiple potential target genes of these miRNAs were closely related to osteogenic differentiation. Importantly, overexpression of miR-146a-5p (an upregulated miRNA) promoted the osteogenic differentiation of hPMSCs, and miR-145-5p overexpression (a downregulated miRNA) inhibited the osteogenic differentiation of hPMSCs.

Synergistic regenerative therapy of thin endometrium by human placenta-derived mesenchymal stem cells encapsulated within hyaluronic acid hydrogels

BackgroundThin endometrium is a primary cause of defective endometrial receptivity, resulting in infertility or recurrent miscarriage. Much effort has been devoted toward regenerating thin endometrium by stem cell-based therapies. The human placenta-derived mesenchymal stem cells (HP-MSCs) are emerging alternative sources of MSCs with various advantages. To maximize their retention inside the uterus, we loaded HP-MSCs with cross-linked hyaluronic acid hydrogel (HA hydrogel) to investigate their therapeutic efficacy and possible underlying mechanisms.MethodsEthanol was injected into the mice uterus to establish the endometrium-injured model. The retention time of HP-MSCs and HA hydrogel was detected by in vivo imaging, while the distribution of HP-MSCs was detected by immunofluorescence staining. Functional restoration of the uterus was assessed by testing embryo implantation rates. The endometrial morphological alteration was observed by H&E staining, Masson staining, and immunohistochemistry. In vitro studies were further conducted using EdU, transwell, tube formation, and western blot assays.ResultsInstilled HP-MSCs with HA hydrogel (HP-MSCs-HA) exhibited a prolonged retention time in mouse uteri than normal HP-MSCs. In vivo studies showed that the HP-MSCs-HA could significantly increase the gland number and endometrial thickness (P < 0.001, P < 0.05), decrease fibrous area (P < 0.0001), and promote the proliferation and angiogenesis of endometrial cells (as indicated by Ki67 and VEGF, P < 0.05, P < 0.05, respectively) in mice injured endometrium. HP-MSCs-HA could also significantly improve the embryo implantation rate (P < 0.01) compared with the ethanol group. Further mechanistic study showed the paracrine effects of HP-MSCs. They could not only promote the proliferation and migration of human endometrial stromal cells via the JNK/Erk1/2-Stat3-VEGF pathway but also facilitate the proliferation of glandular cells via Jak2-Stat5 and c-Fos-VEGF pathway. In turn, the increased VEGF in the endometrium promoted the angiogenesis of endothelial cells.ConclusionOur study suggested the potential therapeutic effects and the underlying mechanisms of HP-MSCs-HA on treating thin endometrium. HA hydrogel could be a preferable delivery method for HP-MSCs, and the strategy represents a promising therapeutic approach against endometrial injury in clinical settings.Graphical abstract

A comparative study of pathogen inactivation technologies in human platelet lysate and its optimal efficiency in human placenta-derived stem cells culture

BackgroundPathogen inactivation (PI) is necessary for the pooled components derived from a biological source. Recently, the use of human platelet lysate (hPL) has increased in the cell manufacturing process as a xeno-free substitute for Fetal Bovine Serum (FBS). Therefore, an effective PI process to produce a pathogen-free hPL with the optimal efficiency in the manufacturing of cell therapy products is a vital requirement. Study design and methodsTo evaluate the efficacy of gamma irradiation and riboflavin/ultraviolet light (RB/UV) as PI methods for hPL, the reduction factor (RF) of titer of model viruses and bacteria were examined. Furthermore, the effect of different PI methods on the hPL performance was evaluated by the in vitro expansion of human placenta-derived mesenchymal stem cells (PLMSCs). To compare different study groups, the growth kinetic, immunophenotype, colony formation, and differentiation capacity (osteogenic and adipogenic) of PLMSCs were examined. In addition, the concentration of growth factors was assayed in each study group. ResultsAchievement to the RF more than 5 log10 for all pathogens, showed the effectiveness of two PI methods. In comparison with the other study groups, the dose of 45 kGy gamma irradiation considerably decreased the growth factor level of the hPL. It also showed a significant adverse effect on PLMSCs growth kinetics. The dose of 30 KGy gamma irradiation and RB/UV demonstrated a favorable effect on different assays of the in vitro expanded PLMSCs. ConclusionThe 30 KGy gamma irradiation and RB/UV were effective in the RF of the viral and bacterial models of the contaminated hPL. The efficacy of these PI-hPLs for PLMSCs expansion was preserved. To increase the safety of cell therapy products, PI methods should be considered for the hPL preparations.

Metabolic Rewiring by Human Placenta-Derived Mesenchymal Stem Cell Therapy Promotes Rejuvenation in Aged Female Rats.

Aging is a degenerative process involving cell function deterioration, leading to altered metabolic pathways, increased metabolite diversity, and dysregulated metabolism. Previously, we reported that human placenta-derived mesenchymal stem cells (hPD-MSCs) have therapeutic effects on ovarian aging. This study aimed to identify hPD-MSC therapy-induced responses at the metabolite and protein levels and serum biomarker(s) of aging and/or rejuvenation. We observed weight loss after hPD-MSC therapy. Importantly, insulin-like growth factor-I (IGF-I), known prolongs healthy life spans, were markedly elevated in serum. Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) analysis identified 176 metabolites, among which the levels of 3-hydroxybutyric acid, glycocholic acid, and taurine, which are associated with health and longevity, were enhanced after hPD-MSC stimulation. Furthermore, after hPD-MSC therapy, the levels of vitamin B6 and its metabolite pyridoxal 5′-phosphate were markedly increased in the serum and liver, respectively. Interestingly, hPD-MSC therapy promoted serotonin production due to increased vitamin B6 metabolism rates. Increased liver serotonin levels after multiple-injection therapy altered the expression of mRNAs and proteins associated with hepatocyte proliferation and mitochondrial biogenesis. Changes in metabolites in circulation after hPD-MSC therapy can be used to identify biomarker(s) of aging and/or rejuvenation. In addition, serotonin is a valuable therapeutic target for reversing aging-associated liver degeneration.

High fibroin-loaded silk-PCL electrospun fiber with core-shell morphology promotes epithelialization with accelerated wound healing.

Silk fibroin (SF) is a widely explored biopolymer for wound-healing applications due to the presence of amino acids in the biodegradable polymer chain with superior mechanical properties. Herein, a high SF-loaded fibrous matrix along with poly(ε-caprolactone) (PCL) was fabricated using electrospinning of emulsion and blend compositions to modulate nanostructure morphology. A comparative study of the physicomechanical properties of electrospun fibers with emulsion (eS7P3) and homogenous blend (bS7P3) was performed as well. In both compositions, SF loading of up to 70% was successfully achieved in the spun fibers while emulsion yielded core-shell morphology, and the blend resulted in monolith fiber architecture as evidenced by TEM microscopy. Further characterization revealed superior mechanical properties in S7P3 fiber with core-shell morphology, as compared to those in the monolith in terms of a higher degree of crystallinity with Young's modulus of 60 MPa under tensile test and nanoindentation modulus of 1.59 ± 0.8 GPa. Further, eS7P3 nanostructure morphology containing silk in the core with a thin outer layer of PCL facilitated relatively faster biodegradation in the lysozyme medium, as compared to that in the monolith. Owing to the presence of a hydrophobic shell, protein adsorption on the fibrous mat presented slow but steady kinetics up to 24 h. When the scaffold was seeded with human placenta-derived mesenchymal stem cells (hPMSCs), in vitro study confirmed that the eS7P3 structure had marginally higher cell proliferation with superior cell infiltration than the monolith. Further, in vivo study involving a rodent model showed the potential of the eS7P3 fiber substrate with a core-shell structure for accelerating full-thickness wound healing by inducing hair follicle and wound closure with less scar formation after 15 days.

Identification of tumorigenic risk genes in human placenta-derived mesenchymal stem cells treated with 3-methylcholanthrene

Mesenchymal stem cells (MSCs) capable of tumour topotaxis have been served as cellular vehicles to deliver anti-tumour agents. As cellular components of the tumour microenvironment, MSCs also affect tumour progression. However, the tumour transformation-related genes of MSCs remain unclear since either tumorigenic or tumour suppressor effects within these cells have been researched. Hence, we aimed to identify potential biomarkers indicative of tumorigenic risk by RNA-seq analysis of human placenta tissue-derived MSCs (hPTMSCs) exposed to the carcinogenic agent, 3-methylcholanthrene (3-MC). Twenty-nine tumour transformation-related genes and three pluripotency-related genes were appraised as differentially expressed genes (DEGs) in hPTMSCs. Overexpression of sfrp1 led to reduced cell viability, migration, and colony formation in A549. In contrast, the overexpression of ptgs2 exerted the opposite effect. These results indicate that A549 cells with high ptgs2 expression but low sfrp1 expression may have a more potential tumorigenic capacity. Taken together, this study suggests that ptgs2 and sfrp1 may be tumorigenic risk genes.

A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

Mesenchymal stem cells (MSCs) have potential role in organ regeneration therapy. Previous work indicating that MSCs confer protection against liver disease. Here, we aimed to determine the potential application in liver regeneration of human placenta-derived MSCs extracellular vesicles (hPMSCs-EVs) via experimental hepatectomy. hPMSCs-EVs were administered intravenously 24 h before 70% partial hepatectomy, the specific composition of hPMSCs-EVs was identified by sequencing and validated by the quantitative polymerase chain reaction, including circ-RBM23. The role of circ-RBM23 in L02 cell was evaluated and it was found that circ-RBM23 knockdown inhibited L02 cell proliferation both in vitro and in vivo. The competing endogenous RNA function of circ-RBM23 was evaluated by the RNA immunoprecipitation assay and found that circ-RBM23 shares miRNA response elements with RRM2. Overexpressed circ-RBM23 bound competitively to miR-139-5p, preventing the miRNA-mediated degradation of RRM2, activating the expression of eIF4G and AKT/mTOR, and facilitating liver regeneration. These results indicate that hPMSCs-EVs prevent hepatic dysfunction and improve liver regeneration in vivo and hepatocytes proliferation in vitro, potentially via circ-RBM23 delivery.

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.

Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model.

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.

Improved wound healing of diabetic foot ulcers using human placenta-derived mesenchymal stem cells in gelatin electrospun nanofibrous scaffolds plus a platelet-rich plasma gel: A randomized clinical trial

AimThe effectiveness of nanofibers containing human placenta-derived mesenchymal stem cells (hPDMSCs) plus platelet-rich plasma (PRP) for healing of diabetic foot ulcers (DFUs) was investigated. MethodshPDMSCs were isolated from human donor placentas, and cultured in electrospun gelatin nanofibrous scaffolds (GNS). Twenty-eight patients with DFUs were randomized into three groups in a 12-week trial: (A) Treated with hPDMSCs; (B) Treated with hPDMSCs after coating the ulcer with PRP gel; (C) Control group received standard wound care. Wound area and pain freewalkingdistance were measured every 2 weeks. ResultsFlow cytometry showed the expression of mesenchymal markers. SEM images and DAPI staining indicated significantly higher levels of hPDMSC proliferation on GNS after 3 and 7 days of culture. The MTS assay showed a significant increase in proliferation on GNS, compared to controls. Wound size reduction was 66% in group A, 71% in group B, and 36% in control group C. A significant difference in wound closure and pain-free walking distance was observed between groups A and B, compared to control group C (p < 0.05), but no difference between groups A and B. Biopsy of the implanted tissue showed the development of new capillary formation in groups A and B. ConclusionImplantation of hPDMSCs in GNS accelerated wound healing and improved clinical parameters in DFU patients.

Comparison on the curative effect of human placenta-derived mesenchymal stem cells and induced islet-like cells in gestational diabetes mellitus rats

Comparison on the curative effect of human placenta-derived mesenchymal stem cells and induced islet-like cells in gestational diabetes mellitus rats

The Protective Benefit of Heme Oxygenase-1 Gene-Modified Human Placenta-Derived Mesenchymal Stem Cells in a N-Nitro-L-Arginine Methyl Ester-Induced Preeclampsia-Like Rat Model: Possible Implications for Placental Angiogenesis.

We previously reported that cytoprotective Heme oxygenase-1, HO-1 (HMOX1) gene-modified human placenta-derived mesenchymal stem cell (HO-1-PMSC) improved placental vascularization in vitro. In the current study, we explored the protective benefit of HO-1-PMSC transplantation in a preeclampsia (PE)-like rat model. A model of PE was successfully constructed by intraperitoneal injection of N-nitro-L-arginine methyl ester (L-NAME). Blood pressure and urinary protein levels were measured. Doppler ultrasound was examined to understand uteroplacental perfusion. ELISA was used to examine the serum levels of VEGF, PlGF, sFlt-1, and sEng. The placentas and fetuses were weighed to verify the improvement in pregnancy outcome. Immunohistochemical and H&E staining was used to detect microvessel density (MVD) in placental tissues and kidney pathology, respectively. The distribution of GFP-labeled PMSC in the placenta were observed under fluorescence microscopy. Blood pressure and proteinuria were reduced and kidney damage was improved. PE rat models treated with PMSC and HO-1-PMSC exhibited an increase in the quality of fetuses and placentas, MVD, VEGF, and PlGF expression, but substantially decreased expression of sFlt-1 and sEng. Doppler ultrasound showed that the placental perfusion was improved. Green fluorescent tracing experiments verified that the cells were successfully transplanted into the placenta and distributed in the blood vessels, indicating that the cells might participate in the process of angiogenesis. These results indicate that therapy with HO-1-PMSC could improve placental vascular dysplasia, increase placental perfusion, control PE symptoms, and promote pregnancy outcome by regulating the balance of angiogenic and antiangiogenic factors or directly participating in the repair of placental vessels in a PE-like rat model.

Rejuvenation of Helicobacter pylori–Associated Atrophic Gastritis Through Concerted Actions of Placenta-Derived Mesenchymal Stem Cells Prevented Gastric Cancer

Chronic Helicobacter pylori infection causes gastric cancer via the progression of precancerous chronic atrophic gastritis (CAG). Therefore, repairing gastric atrophy could be a useful strategy in preventing H. pylori–associated gastric carcinogenesis. Although eradication of the bacterial pathogen offers one solution to this association, this study was designed to evaluate an alternative approach using mesenchymal stem cells to treat CAG and prevent carcinogenesis. Here, we used human placenta-derived mesenchymal stem cells (PD-MSCs) and their conditioned medium (CM) to treat H. pylori–associated CAG in a mice/cell model to explore their therapeutic effects and elucidate their molecular mechanisms. We compared the changes in the fecal microbiomes in response to PD-MSC treatments, and chronic H. pylori–infected mice were given ten treatments with PD-MSCs before being sacrificed for end point assays at around 36 weeks of age. These animals presented with significant reductions in the mean body weights of the control group, which were eradicated following PD-MSC treatment (p < 0.01). Significant changes in various pathological parameters including inflammation, gastric atrophy, erosions/ulcers, and dysplastic changes were noted in the control group (p < 0.01), but these were all significantly reduced in the PD-MSC/CM-treated groups. Lgr5+, Ki-67, H+/K+-ATPase, and Musashi-1 expressions were all significantly increased in the treated animals, while inflammatory mediators, MMP, and apoptotic executors were significantly decreased in the PD-MSC group compared to the control group (p < 0.001). Our model showed that H. pylori–initiated, high-salt diet–promoted gastric atrophic gastritis resulted in significant changes in the fecal microbiome at the phylum/genus level and that PD-MSC/CM interventions facilitated a return to more normal microbial communities. In conclusion, administration of PD-MSCs or their conditioned medium may present a novel rejuvenating agent in preventing the progression of H. pylori–associated premalignant lesions.

Osteogenic ability using porous hydroxyapatite scaffold-based delivery of human placenta-derived mesenchymal stem cells.

Previous preliminary studies have suggested that hydroxyapatite with a grooved structure (HAG) scaffold has good osteogenic potential. This type of scaffold may aid osteogenesis during the repair of large maxillofacial bony defects. The ectopic osteogenic effect and underlying mechanism were further studied using porous HAG scaffold-based delivery of human placenta-derived mesenchymal stem cells (hPMSCs). A total of 18 dogs were randomly allocated into a HAG scaffold group and a HAG scaffold-based hPMSC (HAG/hPMSC) group, and three scaffolds were implanted into the dorsal muscle of each dog. Samples were taken for subsequent analysis and tested 4, 8 and 12 weeks following heterotopic implantation. H&E staining was used to study the osteogenic effect in dog dorsal muscles, and RNA sequencing (RNA-seq) was used for exploring the underlying osteogenic mechanism. The osteogenic ability and effector of the HAG/hPMSC group were significantly greater than those of the HAG scaffold group at 4 weeks after implantation. After 12 weeks, a mature bone plate structure was seen in the HAG/hPMSC group. RNA-seq demonstrated that various osteogenesis-related pathways participated at different stages of metabolism, and that the expression of collagen-1 and runt-related transcription factor 2 increased with implantation time. The present study preliminarily focused on the ectopic osteogenic effect of the porous HAG scaffold-based delivery of hPMSCs in vivo, which may be helpful for the improved application of HAG scaffolds in the future.

MIT-001 Restores Human Placenta-Derived Mesenchymal Stem Cells by Enhancing Mitochondrial Quiescence and Cytoskeletal Organization.

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.

Hypoxia-inducible factor-1α-mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2.

BACKGROUNDAs human placenta-derived mesenchymal stem cells (hP-MSCs) exist in a physiologically hypoxic microenvironment, various studies have focused on the influence of hypoxia. However, the underlying mechanisms remain to be further explored.AIMThe aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODSA hypoxic cell incubator (2.5% O2) was used to mimic a hypoxic microenvironment. Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs. The cell cycle was profiled by flow cytometry. Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing. CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction. Small interfering RNA-mediated hypoxia-inducible factor 1α (HIF-1α) or CD99 knockdown of hP-MSCs, luciferase reporter assays, and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis. Protein expression was assayed by western blotting; immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTSHypoxia enhanced hP-MSC proliferation, increased the expression of cyclin E1, cyclin-dependent kinase 2, and cyclin A2, and decreased the expression of p21. Under hypoxia, CD99 expression was increased by HIF-1α. CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSIONHypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Andrographolide promotes proliferative and osteogenic potentials of human placenta-derived mesenchymal stem cells through the activation of Wnt/\u03b2-catenin signaling

IntroductionThe in vitro expansion and differentiation of mesenchymal stem cells derived from bone marrow (BM-hMSCs) are considered as potential therapeutic tools for clinical applications in bone tissue engineering and regenerative medicine. However, invasive sampling and reduction in number and proliferative capacity with age are the major limitations of BM-hMSCs. Recently, human placenta-derived MSCs (PL-hMSCs) obtained by a non-invasive procedure have attracted much interest. Attempts to increase the potential of PL-hMSCs would be an important paradigm in regenerative medicine. Herein, we examined the proliferative and osteogenic effect of andrographolide (AP) on PL-hMSCs.MethodsMesenchymal stem cells were isolated from full-term normal human placentas and were characterized before using. Cell cytotoxicity and proliferative effect of AP were examined by MTT and BrdU assay, respectively. The non-toxicity concentrations of AP were further assessed for osteogenic effect determined by alkaline phosphatase (ALP) expression and activity, alizarin red staining, and osteoblast-specific gene expressions. Screening of genes involved in osteogenic differentiation-related pathways modulated by AP was explored by a NanoString nCounter analysis.ResultsPL-hMSCs generated in this study met the MSC criteria set by the International Society of Cellular Therapy. The non-cytotoxic concentrations of AP on PL-hMSCs are up to 10 μM. The compound increased PL-hMSC proliferation concomitant with increases in Wnt/β-catenin level and activity. It also enhanced osteogenic differentiation in association with osteoblast-specific mRNA expression. Further, AP promoted bone formation and increased bone structural protein level, osteocalcin, in osteoblastic cells. Gene screening analysis showed the upregulation of genes related to Wnt/β-catenin, TGFβ/BMP, SMAD, and FGF signaling pathways.ConclusionWe demonstrated, for the first time, the potential role of AP in promoting proliferation, osteogenic differentiation, and osteoblast bone formation of PL-hMSCs. This study suggests that AP may be an effective novel agent for the improvement of PL-hMSCs and stem cell-based therapy for bone regeneration.