Advantages of Allogeneic Mesenchymal Stem Cells as an Innovative Therapy in Patients with Endometrium Dysfunction.

Osteoarthritis is a significant and costly cause of pain for both humans and horses. The horse has been identified as a suitable model for human osteoarthritis. Regenerative therapy with allogeneic mesenchymal stem cells (MSCs) is a promising treatment, but the safety of this procedure continues to be debated. The aim of this study is to evaluate the safety of intra-articular injections of allogeneic MSCs on healthy joints by comparing two different dosages and two different tissue sources, namely, bone marrow and umbilical cord blood, with a placebo treatment on the same individuals. We also assessed the influence of autologous versus allogeneic cells for bone marrow-derived MSC treatment. Twelve clinically sound horses were subjected to injections in their 4 fetlock joints. Each of the three fetlocks was administered a different MSC type, and the remaining fetlock was injected with phosphate-buffered saline as a control. Six horses received 10 million cells per joint, and the 6 other horses received 20 million cells per joint. Clinical and ultrasound monitoring revealed that allogeneic bone marrow-derived MSCs induced significantly more synovial effusion compared to umbilical cord blood-derived MSCs but no significant difference was noted within the synovial fluid parameters. The administration of 10 million cells in horses triggered significantly more inflammatory signs than the administration of 20 million cells. Mesenchymal stem cell injections induced mild to moderate local inflammatory signs compared to the placebo, with individual variability in the sensitivity to the same line of MSCs. Understanding the behavior of stem cells when injected alone is a step towards the safer use of new strategies in stem cell therapy, where the use of either MSC secretome or MSCs combined with biomaterials could enhance their viability and metabolic activity.

ObjectiveDiabetes mellitus (DM) is a grave autoimmune disorder because of no insulin self-generation. Currently, mainly clinical methods exist, serious adverse effects leading to stem cell therapy are considered. The mesenchymal stem cells (MSCs), require high differentiation capacity and are judged as crucial in DM treatment. The meta-analysis aimed to systemically analyze the particular types of MSCs which play a more important role in DM and which DM is treated more effectively.MethodA systematic review was conducted on the published literature, clinical trials and observational studies, utilizing databases such as PubMed, Embase, Cochrane and clinicaltrial.gov. RevMan software was adopted to draw Forest Plot and Funnel Plot, and subgroup analysis were employed to evaluate heterogeneity between different groups.ResultsWe identified the meta-analyses of 34 unique random controlled trials and divided our own systematic reviews into 8 groups. The MSCs were associated with placebo (OR = 2.79, 95% CI [1.63, 4.75]), Standard Clinical Treatment (SCT) (OR = 4.12, 95% CI [2.76, 6.14]), and monocyte (OR = 6.52, 95% CI [3.56, 9.48]). The comparison between Autologous MSCs and Allogenic MSCs (OR = 4.64, 95% CI [3.42, 6.31]), Autologous BMMSCs and other MSCs (OR = 5.28, 95% CI [3.64, 7.66]), Allogenic ASCs and UCMSCs (OR = 3.54, 95% CI [1.83, 6.86]), Type I DM and Type II DM (OR = 3.10, 95% CI [1.79, 5.38]), intravenous injection and other injections (OR = 4.81, 95% CI [3.34, 6.94]), diabetic foot ulcers and diabetic neurological disease (OR = 3.88,,95% CI [2.53,5.95]).ConclusionCurrent evidence suggests that MSCs hold significant potential for treating DM, demonstrating considerably high safety and efficacy. MSCs exhibit higher therapeutic benefits compared to monocytes, with autologous MSCs offering better clinical outcomes than allogenic sources. MSCs (BMMSCs) proved more effective than other types of MSCs. However, no significant differences were observed between adipose-derived MSCs (ASCs) and umbilical cord-derived MSCs (UCMSCs) in the allogeneic setting. Moreover, MSCs show more pronounced therapeutic effects in Type II DM, and the difference among the injection methods is minimally observed. In conclusion, the research scope on DM is relatively limited in this study and further research is necessary to improve the reliability of the estimates.

Background Mesenchymal stem cells (MSCs) are known to possess significant immunosuppressive properties, and their use in refractory SLE is supported by promising safety and efficacy in autoimmune animal models and human trials. We conducted this phase I open-label study to test the hypothesis that a single infusion of human allogeneic umbilical cord-derived MSCs (IND 16377) is safe when added to standard-of-care therapy for active SLE. The primary safety outcome is frequency of Grade 3 or higher adverse events (AEs) by Week 24. The primary efficacy outcome is change in SLE disease activity between Baseline and Week 24 measured by SLEDAI score and prednisone dose. Methods Patients (n=6) with active SLE (SLEDAI ≥6) who signed informed consent and met all inclusion and exclusion criteria sequentially received 1 × 106 cells/kg umbilical cord-derived MSCs given as an IV infusion in Plasma-Lyte A solution. Post-infusion, Week 1 and 2 safety data from each participant was reviewed by the Data Safety Monitoring Board prior to enrolling the next patient. Primary safety and efficacy endpoints were determined at Week 24. Each patient is followed for a total of 52 weeks. Results Table 1 summarizes the demographics, visit SLEDAI scores, number of serious and non-serious adverse events (SAEs, AEs). The 6th participant completed their Week 24 visit on April 11, 2018. To date, there has been 1 SAE, deemed not unexpected and not attributed to investigational product. No non-serious AEs higher than Grade 2 by NCI-CTCAE scoring were reported. The SAE was prolonged hospitalization following a partial dose infusion of rituximab IV leading to anaphylaxis. Rituximab was started for persistent SLE disease activity (patient dropped out of the study after Week 8) and was given in the hospital ICU setting due to her prior history of anaphylaxis to Tween (polyethoxylated surfactant found in IV and SQ medications). Anaphylaxis resulted in a prolonged hospital stay of 2 days, resolving with treatment without sequelae. The SAE was attributed to her known allergy to ingredients in the rituximab infusion and deemed unrelated to the MSCs that she received several months earlier. The AEs ‘possibly’ attributable to the investigational product were Grade 2 nausea, Grade 2 tachycardia, and Grade 1 flushing with Grade 1 toe paresthesias – all of which resolved without sequelae. Among the 5 patients who completed their Week 24 evaluations, all showed improved SLE activity (mean SLEDAI score 8.6±1.9 at Baseline improved to 2.6±2.8 at Week 24) with stable or lower doses of prednisone and stable background immunosuppressants. Mean physician global assessment (PGA) scores also improved from 1.71±0.48 at Baseline to 0.32±0.17 at Week 24. Conclusions A single-dose of umbilical cord-derived MSCs was safe and well-tolerated in this open-label phase I trial for 6 patients with active SLE. Initial efficacy data for MSCs in SLE appears promising and will be further tested in a larger controlled trial. Acknowledgements We would like to thank all of the study participants for their time and commitment to the study. This research study was supported by grants from the Lupus Foundation of America and the National Institutes of Health: National Institute of Allergy and Infectious Diseases (R34 AI114453) and the South Carolina Clinical and Translational Research (SCTR) Institute at the Medical University of South Carolina (UL1 RR029881). Trial registration NCT 03171194

Platelet-rich plasma (PRP) has been suggested for the improvement of endometrial growth and receptivity in the patients with recurrent implantation failure (RIF). The aim of present study was to investigate the impact of different concentration of PRP on the expression of genes involved in the endometrial receptivity in the human endometrial cells from RIF and controls with thin and normal endometrium in vitro. In this cross-sectional study, endometrial biopsies were obtained from 14 healthy fertile women and 14 women with RIF. Endometrial cells from 4 different group (RIF and control with endometrial thickness < 7mm and > 7mm) were cultured with three different concentration of PRP 3%, 5% and 10%. Expression of leukemia inhibitory factor (LIF), COX2 and P53, estrogen receptors (ERs) and progesterone receptors (PRs) genes were measured using quantitative real-time polymerase chain reaction (PCR). Protein expression levels of LIF, COX2 and p53 were evaluated using Western Blot method (WB). There was a significant decrease in the expression of PROA/b, ER2/b, LIF/b, COX2/b and P53/b genes in the RIF groups compared to the controls. Treatment with 5% and 10% PRP caused a significant increase in the gene expression of PRs, ERs, LIF/b, COX2/b and p53 in the RIF groups. Moreover, protein expression of COX2/b, LIF/b and p53/b increased following treatment with PRP in the RIF group with the endometrium thickness < 7mm. PRP enhances expression of LIF, COX2, p53, ERs and PRs in the RIF patients with thin endometrium which may improve endometrium receptivity.

Towards a consensus potency assay for mesenchymal stromal cells: a matrix analysis of cell source, donor variability and inflammatory stimuli to refine surrogate markers of immunomodulation

Bone marrow derived allogeneic (donor derived) mesenchymal stem cells (MSCs) are considered to be prominent cell type for cardiac repair following a damage due to ischemic heart disease. Even though the outcome of initial allogeneic MSCs based clinical trials was encouraging, the overall enthusiasm lately has declined due to poor survival of transplanted cells in the ischemic heart. We reported in a rat model of myocardial infarction that allogeneic-MSCs became immunogenic after 5 weeks of transplantation and were rejected by host immune system that led to poor survival of implanted cells. The immunoprivilege of MSCs is preserved by absence of cell surface antigen, human leukocyte antigen (HLA) - DRα. We found that in normoxic MSCs, 26S proteasome degrades HLA-DRα and maintains immunoprivilege of MSCs. The exposure to hypoxia leads to inactivation of 26S proteasome and formation of immunoproteasome in MSCs, which is associated with upregulation and activation of HLA-DRα, and as a result MSCs become immunogenic. Furthermore, inhibition of immunoproteasome formation in hypoxic MSCs preserves the immunoprivilege. Therefore, hypoxia induced shift in the phenotype of proteasome from 26S toward immunoproteasome triggers loss of immunoprivilege of allogeneic MSCs and rejection. In our ongoing studies we are investigating if preventing the formation of immunoproteasome would prevent rejection of allogeneic MSCs in the ischemic heart and improve survival of transplanted cells. The outcome of these studies may provide molecular targets to plan interventions to preserve immunoprivilege of allogeneic MSCs in the ischemic heart and improve benefits of allogeneic MSCs for cardiac repair.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. Objectives: To determine and correlate single-cell UC-MSC transcriptomic profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested invivo in hyperoxia-induced neonatal lung injury in rats. Measurements and Main Results: UC-MSCs showed limited transcriptomic heterogeneity but were different from HNDFs. Gene Ontology enrichment analysis revealed distinct (progenitor-like and fibroblast-like) UC-MSC subpopulations. Only treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of nontherapeutic cells and associated with decreased lung retention. Conclusions: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Regulation of decidualization is decisive for proper implantation and the establishment of pregnancy. Recent studies have suggested that several bone morphogenetic proteins (BMPs) play physiological roles in reproduction. In the present study, we examined the expression of BMP7 in the endometrium and the effect of BMP7 on decidualization and proliferation of endometrial stromal cells (ESC). The gene expression of BMP7 in endometrial tissues collected from women with regular menstrual cycles was determined and the effect of ovarian steroid hormones on BMP7 gene expression was investigated in cultured ESC. The effect of BMP7 on the decidualization of ESC was determined by measuring the gene expression and protein secretion of insulin-like growth factor binding protein 1 (IGFBP1), a marker of decidualization. The effect of BMP7 on the proliferation of ESC was examined by the bromodeoxyuridine (BrdU) incorporation assay. The gene expression of BMP7 in endometrial tissues was low at and after the mid-secretory phase of the menstrual cycle. Progesterone suppressed the gene expression of BMP7 in cultured ESC. Treatment with progesterone and estradiol for 12 days achieved decidualization of ESC, increasing the gene expression and protein secretion of IGFBP1. Addition of BMP7 protein to the culture almost completely inhibited these increases. BMP7 suppressed BrdU incorporation in ESC, which indicated an antiproliferative effect of BMP7 on ESC. Progesterone-induced suppression of BMP7 and BMP7-induced inhibition of decidualization and proliferation of ESC suggest an elaborate regulatory mechanism for decidualization through BMP7 in the endometrium.

Allogeneic (donor derived) mesenchymal stem cells (MSCs) derived from bone marrow are in phase I and II clinical trials for cardiac regeneration. Even though the outcome of allogeneic MSCs based animal studies and initial clinical trials was encouraging, the overall enthusiasm of lately has come down. This is due to poor survival of transplanted cells in the recipient heart. The recent reports on allogeneic MSCs based studies found a switch in the phenotype of transplanted cells from immunoprivileged to immunogenic state that led to rejection of cells by host immune system. In the current study, we discovered a novel mechanism of immune switch in MSCs. Our studies demonstrate that hypoxia/or ischemic environment induces an immune shift in MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by absence of major histocompatibility complex class II (MHC‐II) molecules. We found that 26S proteasome‐mediated degradation of MHC‐II prevents its expression on cell surface in MSCs and preserves their immunoprivilege. The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC‐II, and increased immunogenicity of MSCs. Furthermore, hypoxia‐induced downregulation of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs prevented 26S inactivation and preserved the immunoprivilege of MSCs. Therefore, hypoxia‐induced defects in 26S proteasome assembly causes loss of immunoprivilege of allogeneic MSCs. Maintaining 26S proteasome activity in MSCs preserves immunoprivilege and prevents rejection of allogeneic stem cells in the heart.Support or Funding InformationCanadian Institute of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recurrent implantation failure (RIF) patients exhibit poor endometrial receptivity and abnormal decidualization with reduced effectiveness and exposure to progesterone, which is an intractable clinical problem. However, the associated molecular mechanisms remain elusive. We found that EH domain containing 1 (EHD1) expression was abnormally elevated in RIF and linked to aberrant endometrial decidualization. Here we show that EHD1 overexpressed in human endometrial stromal cells significantly inhibited progesterone receptor (PGR) transcriptional activity and the responsiveness to progesterone. No significant changes were observed in PGR mRNA levels, while a significant decrease in progesterone receptor B (PRB) protein level. Indeed, EHD1 binds to the PRB protein, with the K388 site crucial for this interaction. Overexpression of EHD1 promotes the SUMOylation and ubiquitination of PRB, leading to the degradation of the PRB protein. Supplementation with the de-SUMOylated protease SENP1 ameliorated EHD1-repressed PRB transcriptional activity. To establish a functional link between EHD1 and the PGR signalling pathway, sg-EHD1 were utilized to suppress EHD1 expression in HESCs from RIF patients. A significant increase in the expression of prolactin and insulin-like growth factor-binding protein 1 was detected by interfering with the EHD1. In conclusion, we demonstrated that abnormally high expression of EHD1 in endometrial stromal cells attenuated the activity of PRB associated with progesterone resistance in a subset of women with RIF.

Umbilical cord derived mesenchymal stem cells attenuate rodent Osteoarthritis progression via preserving articular cartilage superficial layer cells and inhibiting synovitis

In a mouse model of menstruation stromal cells respond to breakdown of the tissue by changing their identity to become epithelial cells that are incorporated into the luminal epithelium which is rapidly 'healed' without scarring.

To evaluate the impact of autologous platelet-rich plasma (PRP) intrauterine perfusion on endometrial receptivity and pregnancy outcomes in patients with implantation failure. This retrospective cohort study included patients who visited our hospital between January 2019 and January 2024, all suffering from recurrent implantation failures and diagnosed with thin endometrium. Participants were divided into two groups: conventional-treatment (n=116) and PRP-treatment (n=104). Serum levels of estradiol 2 (E2), follicle stimulating hormone (FSH), and luteinizing hormone (LH) were measured before and 14 days after treatment. Transvaginal Doppler ultrasound assessed uterine artery, arcuate artery, and spiral artery pulsatility index (PI) and resistance index (RI) at both timepoints. Endometrial thickness was measured before and 14 days post-treatment. Pregnancy outcomes, including implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate, were recorded. The PRP-treatment group showed a significant increase in endometrial thickness (P=0.037). Uterine artery PI (P=0.004) and RI (P=0.015), arcuate artery PI (P=0.037) and RI (P=0.003), and spiral artery PI (P=0.002) significantly decreased in the PRP-treatment group compared to the conventional-treatment group. Hormonal measurements indicated higher E2 (P=0.001) and lower FSH (P=0.002) and LH (P=0.002) levels post-treatment in the PRP-treatment group. Pregnancy outcomes significantly favored the PRP-treatment group, with higher implantation rates (P=0.004), clinical pregnancy rates (P=0.044), ongoing pregnancy rates (P=0.031), and live birth rates (P=0.032). PRP intrauterine perfusion significantly improves endometrial parameters, uterine hemodynamics, and pregnancy outcomes in patients with recurrent implantation failure and thin endometrium.

Background: Mesenchymal stem cells (MSCs) exhibit a high capacity for differentiation, possess anti-inflammatory and proangiogenic properties, and stimulate the growth and proliferation of neighboring cells. MSCs are a promising tool in regenerative medicine. However, the molecular mechanisms underlying the properties of these cells are not yet fully understood. Gene expression in MSCs influences their characteristics and differentiation potential. Therefore, it is essential to investigate factors affecting gene expression as well as those activating signaling pathways, which will enable more effective and individualized applications of MSCs. In this study, we aimed to identify signaling pathways involved in gene expression in umbilical cord-derived MSCs (UC-MSCs) that may be altered by maternal diabetes and hypothyroidism during pregnancy. Methods: The research material consisted of UC-MSCs. Samples obtained from nine participants were analyzed. UC-MSCs were isolated and cultured, and RNA was extracted. The isolated RNA was used for microarray-based gene expression analysis. Subsequently, pathway enrichment analysis was performed to identify the signaling pathways involved. Results: In the diabetes group, 340 genes (0.71%) were upregulated, while 268 genes (0.56%) were downregulated compared with UC-MSCs from the control group. In the diabetes group, the most compact module was composed of proteins associated with WNT/planar cell polarity (WNT/PCP) signaling. The second module included genes related to smooth muscle activity. In the hypothyroidism group, an association was identified between the extracellular matrix organization pathways (GO:0030198) and the extracellular structure organization (GO:0043062) pathways. Moreover, in this group, increased expression of MMP1, MMP10, and GREM1 was observed. Conclusions: In summary, our study demonstrated the impact of diabetes and hypothyroidism on gene expression in UC-MSCs. We also observed the activation of distinct signaling pathways depending on the presence of these conditions. However, this work represents a preliminary screening, and the results should be validated by PCR in a larger cohort.

Mesenchymal stem cells (MSCs) have attracted significant attention recently because of their immunomodulatory function. Due to their immunosuppressive role, MSCs have been suggested as a novel medical therapeutic option to treat graft-versus-host disease (GVHD) and autoimmune diseases. Severe GVHD is a lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, there have been only a few limited therapeutic options for the treatment of GVHD. Since the initial studies and use of MSCs, density-gradient centrifugation-based isolation has been the primary conventional protocol for the isolation of clinical trial grade nonclonal MSCs in the treatment of severe GVHD and other diseases. Recently, clonal MSCs isolated by the subfractionation culturing method (SCM) have been also used in a clinical setting to treat severe GVHD. Although there are concerns about the immunogenicity of allogeneic MSCs, all the clinical trials using allogeneic nonclonal and clonal MSCs performed in the last decade have shown that no significant adverse effects or ectopic transformation were involved with allogeneic MSCs. This suggests that allogeneic MSCs can be a promising therapeutic option for the treatment of steroid- refractory acute GVHD. However, further investigations are needed to understand the exact mechanisms of the functions of MSCs and larger, well-designed human clinical trials are necessary to confirm the long-term safety and efficacy of MSCs for the treatment of GVHD and other autoimmune diseases.