Optimization of an Affordable and Efficient Skin Allograft Composite with Excellent Biomechanical and Biological Properties Suitable for the Regeneration of Deep Skin Wounds: A Preclinical Study.

Deep skin wounds require grafting with a skin substitute for treatment. Despite many attempts in the development of an affordable and efficient skin substitute, the repair of deep skin wounds still remains challenging. In the current study, we present a 3D sponge composite made from human placenta (a disposable organ) and sodium alginate with exceptional properties for skin tissue engineering applications. Toward this goal, different proportions of alginate (Alg) and decellularized placenta scaffold (DPS) were composited and freeze-dried to generate a 3D sponge with the desired biomechanical and biological features. Comprehensive in vitro, in ovo, and in vivo characterizations were performed to assess the morphology, physical structure, mechanical behaviors, angiogenic potential, and wound healing properties of the composites. Through these analyses, the scaffold with optimal proportions of Alg (50%) and DPS (50%) was found to have superior properties. The optimized scaffold (Alg50/DPS50) was applied to the full-thickness wounds created in rats. Our data revealed that the addition of DPS to the Alg solution caused a significant improvement in the mechanical characteristics of the scaffold. Remarkably, the fabricated composite scaffold exhibited mechanical properties similar to those of native skin tissue. When implanted into the full-thickness wounds, the Alg50/DPS50 composite scaffold promoted angiogenesis, re-epithelialization, and granulation tissue formation, as compared to the group without a scaffold. Overall, our findings underscore the potential value of this hybrid scaffold for enhancing skin wound healing and suggest an Alg50/DPS50 composite for clinical investigations.

Wharton's Jelly mesenchymal stem cell-derived extracellular vesicles induce liver fibrosis-resolving phenotype in alternatively activated macrophages.

The potential of extracellular vesicles (EVs) isolated from mesenchymal stromal cells in guiding macrophages toward anti-inflammatory immunophenotypes, has been reported in several studies. In our study, we provided experimental evidence of a distinctive effect played by Wharton Jelly mesenchymal stromal cell-derived EVs (WJ-EVs) on human macrophages. We particularly analyzed their anti-inflammatory effects on macrophages by evaluating their interactions with stellate cells, and their protective role in liver fibrosis. A three-step gradient method was used to isolate monocytes from umbilical cord blood (UCB). Two subpopulations of WJ-EVs were isolated by high-speed (20,000 g) and differential ultracentrifugation (110,000 g). Further to their characterization, they were designated as EV20K and EV110K and incubated at different concentrations with UCB-derived monocytes for 7 days. Their anti-fibrotic effect was assessed by studying the differentiation and functional levels of generated macrophages and their potential to modulate the survival and activity of LX2 stellate cells. The EV20K triggers the polarization of UCB-derived monocytes towards a peculiar M2-like functional phenotype more effectively than the M-CSF positive control. The EV20K treated macrophages were characterized by a higher expression of scavenger receptors, increased phagocytic capacity and production level of interleukin-10 and transforming growth factor-ß. Conditioned medium from those polarized macrophages attenuated the proliferation, contractility and activation of LX2 stellate cells. Our data show that EV20K derived from WJ-MSCs induces activated macrophages to suppress immune responses and potentially play a protective role in the pathogenesis of liver fibrosis by directly inhibiting HSC's activation.

The Application of Umbilical Cord Blood-derived Platelet Gel for Skin Ulcers Associated With Chronic Graft-Versus-Host Disease in Pediatrics: A Randomized Trial.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative strategy against a variety of malignant and nonmalignant disorders. However, acute and chronic graft-versus-host disease (aGVHD and cGVHD, respectively) commonly complicate this approach, culminating in substantial morbidities and mortalities. The integumentary system is the preponderant organ involved in cGVHD, and its response to existing treatments, including well-versed immunosuppressants and novel targeted therapies, is not desirable. Despite the rarity, ulcers of sclerotic skin cGVHD are treatment-refractory and associated with significant morbidities and an exaggerated risk of infectious complications. Platelet-rich plasma (PRP) and its derivatives are endowed with growth factors and proangiogenic molecules and hold regenerative potential. This study aimed to assess the safety and efficacy of the application of platelet gel-containing dressing against ulcerative skin cGVHD in pediatric patients. This randomized trial is conducted at the hematopoietic stem cell transplantation unit of the Children's Medical Center Hospital in Tehran, Iran. Twenty-one pediatric patients (aged between 5 and 15 years) were initially enrolled, and 16 met the inclusion criteria. All cases (4 females) were recipients of allo-HSCT who had been complicated with symmetrically or near-symmetrically ulcerative sclerotic skin cGVHD. Fresh umbilical cord blood (UCB) was obtained from healthy donors and underwent centrifugation using a novel PRP preparation kit in a single-step process. Platelet gel was produced by adding thrombin to the isolated buffy coat layer. Two similar ulcers of each patient were randomized to receive either conventional dressing or platelet gels up to 6 times. At each time point evaluation, ulcer size and its relative reduction compared to the basal size were recorded. Included patients received a total of 80 platelet gel-containing dressings. While the mean sizes of randomized ulcers at the beginning of the study were similar, their differences became significant 15 days after the initiation of intervention (P = .019). In addition, the mean reduction in the ulcers' surface area (in comparison to their baseline values) was significantly higher for the intervention arm at all evaluation points (P = .001 for day 5 and P < .001 for subsequent time points). At the end of the trial, the number of ulcers with a more than 50% reduction in size was 14 (87.5%) in the intervention arm (including 6 completely healed ulcers) versus 1 (6.25%, which was not completely healed) in the control arm (P < .001). None of the patients exhibited any localized or systemic treatment-related adverse events. In this study, using a relatively large number of cases, we showed that UCB-derived platelet gel is a safe, feasible, and effective curative approach for skin ulcers of sclerotic skin cGVHD in pediatric patients. Designing upcoming trials on the efficacy of this therapeutic approach for ocular, mucosal, and acute skin GVHD is prudent. Retrospectively registered at the Iranian Registry of Clinical Trials (registration number IRCT20190101042197N1) on August 24, 2020.

Human Umbilical Cord Mesenchymal Stem Cell-Derived Microvesicles Could Induce Apoptosis and Autophagy in Acute Myeloid Leukemia.

Background: Microvesicles (MV) have been identified as candidate biomarkers for treating acute myeloid leukemia (AML). This study investigated the effects of human umbilical cord-derived mesenchymal stem cell (hUCMSC)-derived MVs on apoptosis and autophagy in the KG-1 leukemic cell line. Methods: The hUCMSCs were cultured and characterized by flow cytometry. MVs were isolated by ultracentrifugation, and the concentration was determined using the Bradford method. The characteristics of MVs were confirmed using transmission electron microscopy, flow cytometry, and dynamic light scattering methods. KG-1 cells were treated with the desired concentrations of MVs for 24 h. The apoptosis induction and reactive oxygen species production were evaluated using flow cytometry. RT-PCR was performed to evaluate apoptosis- and autophagy-related genes expression. Results: Following tretment of KG-1 cells with 25, 50, and 100 µg/ml concentrations of MVs, the apoptosis rates were 47.85%, 47.15%, and 51.35% (p < 0.0001), and the autophagy-induced ROS levels were 73.9% (p < 0.0002), 84.8% (p < 0.0001), and 85.4% (p < 0.0001), respectively. BAX and ATG7 gene expression increased significantly at all concentrations compared to the control, and this level was higher at 50 µg/ml than that of the other concentrations. In addition, LC3 and Beclin 1 expression increased significantly in a concentration-dependen manner. Conversely, BCL2 expression decreased compared to the control. Conclusion: Our findings indicate that hUCMSC-MVs could induce cell death pathways of autophagy and apoptosis in the KG-1 cell lines and exert potent antiproliferative and proapoptotic effects on KG-1 cells in vitro. Therefore, hUCMSC-MVs may be a potential approach for cancer therapy as a novel cell-to-cell communication strategy.

The Association of Human Leucocyte Antigen (HLA) Class I and II Genes with Cutaneous and Visceral Leishmaniasis in Iranian Patients: A Preliminary Case-Control Study.

Background: Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Methods: Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. Results: There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P<0.05). Conclusion: This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Umbilical Cord Blood-Derived Monocytes as A Reliable Source of Functional Macrophages for Biomedical Research.

OBJECTIVE: Macrophages are multifunctional immune cells widely used in immunological research. While autologous macrophages have been widely used in several biomedical applications, allogeneic macrophages have also demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure monocytes with the potential to differentiate into functional macrophages. MATERIALS AND METHODS: In the experimental study, we describe a simple and efficient protocol to isolate highpurity monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well. RESULTS: A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity. CONCLUSION: Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for biomedical research.

Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial.

BACKGROUND AND AIMS: The main causes of death in patients with severe Coronavirus disease-2019 (COVID-19) are acute respiratory distress syndrome (ARDS) and multiorgan failure caused by a severe inflammatory cascade. Novel treatment strategies, such as stem-cell-based therapy and their derivatives can be used to relieve inflammation in these cases. In this study, we aimed to evaluate the safety and efficacy of therapy using mesenchymal stromal cells (MSCs) and their derived extracellular vesicles in COVID-19 patients. MATERIALS AND METHODS: COVID-19 patients with ARDS were included in this study and allocated into two study and control groups using block randomization. While all patients received recommended treatment based on guidelines from the national advisory committee for COVID-19 pandemic, the two intervention groups received two consecutive injections of MSCs (100 × 106 cells) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived extracellular vesicles (EVs). Patients were assessed for safety and efficacy by evaluating clinical symptoms, laboratory parameters, and inflammatory markers at baseline and 48 h after the second intervention. RESULTS: A total number of 43 patients (the MSC alone group = 11, MSC plus EV group = 8, and control group = 24) were included in the final analysis. Mortality was reported in three patients in the MSC alone group (RR: 0.49; 95% CI 0.14-1.11; P = 0.08); zero patient in the MSC plus EV group (RR: 0.08; 95% CI 0.005-1.26; P = 0.07) and eight patients in the control group. MSC infusion was associated with a decrease in inflammatory cytokines such as IL-6 (P = 0.015), TNF-α (P = 0.034), IFN-γ (P = 0.024), and CRP (P = 0.041). CONCLUSION: MSCs and their extracellular vesicles can significantly reduce the serum levels of inflammatory markers in COVID-19 patients, with no serious adverse events. Trial registration IRCT, IRCT registration number: IRCT20200217046526N2. Registered 13th April 2020, http://www.irct.ir/trial/47073 .

Platelet-Rich Plasma in Regenerative Medicine: Possible Applications in Management of Burns and Post-Burn Scars: A Review.

Contribution of platelets in tissue regeneration and their possible application in regenerative medicine, which is primarily mediated via secretion of granular components following platelet activation, has been well established in the recent decades. Therefore, platelet rich plasma (PRP), as a portion of plasma with higher concentrations of platelets than the baseline level, is now an attractive therapeutic option in various medical fields mainly for tissue repair and regeneration following injuries. Burn injuries are devastating trauma with high rate of morbidities affecting several aspects of the patient's life. They require a long-time medical care and high costs. However, even following the best treatment procedures, post-burn scars are inevitable consequence of burn healing process. Therefore, development of new treatment modalities for both burn healing and prevention of post-burn scar establishment seems to be necessary. Regarding the well-known role of PRP in wound healing, here we aimed to provide a comprehensive insight in the possible application of PRP as an adjuvant therapy for the management of burn injuries and subsequent scars. In terms of the following keywords (individually or in combination), original/review articles were searched in PubMed, Scopus, and Google Scholar databases from 2009 to 2021: platelet rich plasma, PRP therapy, platelet biology, platelet function, burn healing, burn scar, scar formation, burn management, wound healing, regenerative medicine. All type of articles or book chapters in English language and relevant data were included in this review. This review initially focused on PRP, its mechanisms of action, preparation methods, and available sources. Then, pathophysiology of burns and subsequent scars were discussed. Finally, their current conventional therapeutic modalities and implication of PRP in their healing process were highlighted.

Protective effect of human amniotic membrane extract against hydrogen peroxide-induced oxidative damage in human dermal fibroblasts.

OBJECTIVE: One of the main approaches to preventing skin ageing is to protect fibroblast cells from oxidative stress. The promoting effect of the human amniotic membrane extract (hAME) on re-epithelization, proliferation and migration of cells in wound healing has been already well studied. This experimental study aimed to investigate the antioxidant activity of hAME against hydrogen peroxide (H2 O2 )-induced dermal fibroblast damage. METHODS: Here, to establish the ageing model, human foreskin fibroblasts (HFFs) were exposed to 200 µM H2 O2 for 2 h. HFFs were treated with 0.1 mg/ml AME for 24 or 48 h before or/and after H2 O2 exposure. A total of 48 h following the H2 O2 treatment, we measured cell proliferation, viability, senescence-associated ß-galactosidase (SA-ß-Gal), antioxidants and preinflammatory cytokine (IL-6) levels, as well as the expression of senescence-associated genes (P53 and P21). RESULTS: The obtained results indicated that under oxidative stress, AME significantly increased cellular viability and not only promoted the cell proliferation rate but also attenuated apoptotic induction condition (p < 0.001). AME also significantly reversed the SA-ß-Gal levels induced by H2 O2 (p < 0.001). Additionally, both pre- and post-treatment regimen by AME down-regulated the expression of senescence marker genes (p < 0.001). Moreover, AME declined different oxidative stress biomarkers such as superoxide dismutase and catalase and increased the glutathione amount. CONCLUSION: Altogether, our results indicated that AME had a remarkable antioxidant and antiageing activity as pre- and post-treatment regimen, pointing to this compound as a potential natural-based cosmeceutical agent to prevent and treat skin ageing conditions.

Human umbilical cord serum as an alternative to fetal bovine serum for in vitro expansion of umbilical cord mesenchymal stromal cells.

In the use of bovine fetal serum (FBS) there is concern about the possibility of disease transmission from animal to human. Therefore, it seems necessary to create culture conditions free of animal serum, especially in cell therapy. The aim of this study was to evaluate the feasibility of replacing human umbilical cord serum (hUCS) with FBS for in vitro expansion of umbilical cord mesenchymal stromal/stem cells (UC-MSCs). Here, UC-MSCs were cultured for five days in media supplemented either by hUCS or commercial FBS (Gibco and HyClone) to compare their viability, proliferation, morphology, Immunophenotype and differentiation potential. Our data shows that use of 5% and/or 10% hUCS, resulted in a tenfold increase in the number of MSCs; While in the presence of commercial FBS, this figure reached a maximum of five times. Notably, the rate of cell proliferation in the group containing 2% hUCS was the same as the groups containing 10% commercial FBS. Furthermore, there was no significant difference between groups in terms of viability, surface markers, and multilineage differentiation potential. These results demonstrated that hUCS can efficiently replace FBS for the routine culture of MSCs and can be used ideally in manufacturing process of UC-MSCs in cell therapy industry.

Extraembryonic Mesenchymal Stromal/Stem Cells in Liver Diseases: A Critical Revision of Promising Advanced Therapy Medicinal Products.

Liver disorders have been increasing globally in recent years. These diseases are associated with high morbidity and mortality rates and impose high care costs on the health system. Acute liver failure, chronic and congenital liver diseases, as well as hepatocellular carcinoma have been limitedly treated by whole organ transplantation so far. But novel treatments for liver disorders using cell-based approaches have emerged in recent years. Extra-embryonic tissues, including umbilical cord, amnion membrane, and chorion plate, contain multipotent stem cells. The pre-sent manuscript discusses potential application of extraembryonic mesenchymal stromal/stem cells, focusing on the management of liver diseases. Extra-embryonic MSC are characterized by robust and constitutive anti-inflammatory and anti-fibrotic properties, indicating as therapeutic agents for inflammatory conditions such as liver fibrosis or advanced cirrhosis, as well as chronic inflammatory settings or deranged immune responses.

The safety and efficacy of umbilical cord blood mononuclear cells in individuals with spastic cerebral palsy: a randomized double-blind sham-controlled clinical trial.

INTRODUCTION: The current multi-center, randomized, double-blind study was conducted among children with cerebral palsy (CP) to assess the safety and efficacy of umbilical cord blood mononuclear cell (UCB-MNC). We performed the diffusion tensor imaging to assess the changes in the white matter structure. METHODS: Males and females aged 4 to 14 years old with spastic CP were included. Eligible participants were allocated in 4:1 ratio to be in the experimental or control groups; respectively. Individuals who were assigned in UCB-MNC group were tested for human leukocyte antigen (HLA) and fully-matched individuals were treated with UCB-MNCs. A single dose (5 × 106 /kg) UCB-MNCs were administered via intrathecal route in experimental group. The changes in gross motor function measure (GMFM)-66 from baseline to one year after treatment were the primary endpoints. The mean changes in modified Ashworth scale (MAS), pediatric evaluation of disability inventory (PEDI), and CP quality of life (CP-QoL) were also evaluated and compared between groups. The mean changes in fractional anisotropy (FA) and mean diffusivity (MD) of corticospinal tract (CST) and posterior thalamic radiation (PTR) were the secondary endpoints. Adverse events were safety endpoint. RESULTS: There were 72 included individuals (36 cases in each group). The mean GMFM-66 scores increased in experimental group; compared to baseline (+ 9.62; 95%CI: 6.75, 12.49) and control arm (ß: 7.10; 95%CI: 2.08, 12.76; Cohen's d: 0.62) and mean MAS reduced in individuals treated with UCB-MNCs compared to the baseline (-0.87; 95%CI: -1.2, -0.54) and control group (ß: -0.58; 95%CI: -1.18, -0.11; Cohen's d: 0.36). The mean PEDI scores and mean CP-QoL scores in two domains were higher in the experimental group compared to the control. The imaging data indicated that mean FA increased and MD decreased in participants of UCB-MNC group indicating improvements in white matter structure. Lower back pain, headaches, and irritability were the most common adverse events within 24 h of treatment that were related to lumbar puncture. No side effects were observed during follow-up. CONCLUSIONS: This trial showed that intrathecal injection of UCB-MNCs were safe and effective in children with CP. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov ( NCT03795974 ).

Clinical and imaging outcomes after intrathecal injection of umbilical cord tissue mesenchymal stem cells in cerebral palsy: a randomized double-blind sham-controlled clinical trial.

BACKGROUND: This study assessed the safety and efficacy of intrathecal injection of umbilical cord tissue mesenchymal stem cells (UCT-MSC) in individuals with cerebral palsy (CP). The diffusion tensor imaging (DTI) was performed to evaluate the alterations in white-matter integrity. METHODS: Participants (4-14 years old) with spastic CP were assigned in 1:1 ratio to receive either UCT-MSC or sham procedure. Single-dose (2 × 107) cells were administered in the experimental group. Small needle pricks to the lower back were performed in the sham-control arm. All individuals were sedated to prevent awareness. The primary endpoints were the mean changes in gross motor function measure (GMFM)-66 from baseline to 12 months after procedures. The mean changes in the modified Ashworth scale (MAS), pediatric evaluation of disability inventory (PEDI), and CP quality of life (CP-QoL) were also assessed. Secondary endpoints were the mean changes in fractional anisotropy (FA) and mean diffusivity (MD) of corticospinal tract (CST) and posterior thalamic radiation (PTR). RESULTS: There were 36 participants in each group. The mean GMFM-66 scores after 12 months of intervention were significantly higher in the UCT-MSC group compared to baseline (10.65; 95%CI 5.39, 15.91) and control (ß 8.07; 95%CI 1.62, 14.52; Cohen's d 0.92). The increase was also seen in total PEDI scores (vs baseline 8.53; 95%CI 4.98, 12.08; vs control: ß 6.87; 95%CI 1.52, 12.21; Cohen's d 0.70). The mean change in MAS scores after 12 months of cell injection reduced compared to baseline (-1.0; 95%CI -1.31, -0.69) and control (ß -0.72; 95%CI -1.18, -0.26; Cohen's d 0.76). Regarding CP-QoL, mean changes in domains including friends and family, participation in activities, and communication were higher than the control group with a large effect size. The DTI analysis in the experimental group showed that mean FA increased (CST 0.032; 95%CI 0.02, 0.03. PTR 0.024; 95%CI 0.020, 0.028) and MD decreased (CST -0.035 × 10-3; 95%CI -0.04 × 10-3, -0.02 × 10-3. PTR -0.045 × 10-3; 95%CI -0.05 × 10-3, -0.03 × 10-3); compared to baseline. The mean changes were significantly higher than the control group. CONCLUSIONS: The UCT-MSC transplantation was safe and may improve the clinical and imaging outcomes. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov ( NCT03795974 ).

Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates.

OBJECTIVE: In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. MATERIALS AND METHODS: In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H and E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. RESULTS: A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). CONCLUSION: It seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Mesenchymal stem cells derived from perinatal tissues for treatment of critically ill COVID-19-induced ARDS patients: a case series.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a fatal complication of coronavirus disease 2019 (COVID-19). There are a few reports of allogeneic human mesenchymal stem cells (MSCs) as a potential treatment for ARDS. In this phase 1 clinical trial, we present the safety, feasibility, and tolerability of the multiple infusions of high dose MSCs, which originated from the placenta and umbilical cord, in critically ill COVID-19-induced ARDS patients. METHODS: A total of 11 patients diagnosed with COVID-19-induced ARDS who were admitted to the intensive care units (ICUs) of two hospitals enrolled in this study. The patients were critically ill with severe hypoxemia and required mechanical ventilation. The patients received three intravenous infusions (200 × 106 cells) every other day for a total of 600 × 106 human umbilical cord MSCs (UC-MSCs; 6 cases) or placental MSCs (PL-MSCs; 5 cases). FINDINGS: There were eight men and three women who were 42 to 66 years of age. Of these, six (55%) patients had comorbidities of diabetes, hypertension, chronic lymphocytic leukemia (CLL), and cardiomyopathy (CMP). There were no serious adverse events reported 24-48 h after the cell infusions. We observed reduced dyspnea and increased SpO2 within 48-96 h after the first infusion in seven patients. Of these seven patients, five were discharged from the ICU within 2-7 days (average: 4 days), one patient who had signs of acute renal and hepatic failure was discharged from the ICU on day 18, and the last patient suddenly developed cardiac arrest on day 7 of the cell infusion. Significant reductions in serum levels of tumor necrosis factor-alpha (TNF-α; P < 0.01), IL-8 (P < 0.05), and C-reactive protein (CRP) (P < 0.01) were seen in all six survivors. IL-6 levels decreased in five (P = 0.06) patients and interferon gamma (IFN-γ) levels decreased in four (P = 0.14) patients. Four patients who had signs of multi-organ failure or sepsis died in 5-19 days (average: 10 days) after the first MSC infusion. A low percentage of lymphocytes (< 10%) and leukocytosis were associated with poor outcome (P = 0.02). All six survivors were well with no complaints of dyspnea on day 60 post-infusion. Radiological parameters of the lung computed tomography (CT) scans showed remarkable signs of recovery. INTERPRETATION: We suggest that multiple infusions of high dose allogeneic prenatal MSCs are safe and can rapidly improve respiratory distress and reduce inflammatory biomarkers in some critically ill COVID-19-induced ARDS cases. Patients that develop sepsis or multi-organ failure may not be good candidates for stem cell therapy. Large randomized multicenter clinical trials are needed to discern the exact therapeutic potentials of MSC in COVID-19-induced ARDS.

Stem Cell Therapy: A Promising Approach in Treatment of COVID-19.

COVID-19 pandemic is a global health crisis of the 21st Century. There are currently no approved vaccines and no particular anti-viral treatment for coronavirus disease. As COVID-19 has a broad range of illnesses, it is necessary to find a safe and effective therapeutic method for COVID-19. An attractive approach for treating COVID-19 is cell therapy. Cell therapy aims to inject new and healthy stem cells into a patient's body, to repair the damaged cells and tissues. Stem cell therapy is one of the most studied and important approaches in the treatment of COVID-19 these days. The significant clinical outcome was observed by the adoptive transfer of stem cells, specifically mesenchymal stem cells. This study reviews the characteristics of stem cells and clinical trials that have used stem cells in treating COVID-19.

Combination of SB431542, Chir9901, and Bpv as a novel supplement in the culture of umbilical cord blood hematopoietic stem cells.

BACKGROUND: Small molecule compounds have been well recognized for their promising power in the generation, expansion, and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood-derived CD34+ cells. METHODS: Considering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pµ, and NAM. The eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell-related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model. RESULTS: Our data shows that the simultaneous use of SB431542 (TGF-ß inhibitor), Chir9901 (GSK3 inhibitor), and Bpv (PTEN inhibitor) resulted in a 50-fold increase in the number of CD34+CD38- cells. This was further reflected in approximately 3 times the increase in the clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of the NMRI model of in utero transplantation. These results are in total conformity with the upregulation of HOXB4, GATA2, and CD34 marker gene as well as the CXCR4 homing gene. CONCLUSION: Taken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.

Assessment of Short-Term Engraftment Potential of Ex Vivo Expanded Hematopoietic Stem Cells Using Normal Fetal Mouse in Utero Transplantation Model.

OBJECTIVE: Ex vivo expansion is a promising strategy to overcome the low number of human umbilical cord blood hematopoietic stem cells (hUCB-HSCs). Although based on the obtained results in unnatural physiological condition of irradiated genetically immune-deficient mouse models, there has always been concern that the expanded cells have less engraftment potential. The purpose of this study was to investigate effect of common ex vivo expansion method on engraftment potential of hUCB-mononuclear cells (MNCs), using normal fetal mouse, as a model with more similarity to human physiological conditions. MATERIALS AND METHODS: In this experimental study, briefly, isolated hUCB-MNCs were cultured in common expansion medium containing stem cell factor, Flt3 ligand and thrombopoietin. The unexpanded and expanded cells were transplanted to the fetal mice on gestational days of 11.5-13.5. After administration of human hematopoiesis growth factors (hHGFs), presence of human CD45+ cells, in the peripheral blood of recipients, was assessed at various time points after transplantation. RESULTS: The expanded MNCs showed 32-fold increase in the expression of CD34+38- phenotype and about 3-fold higher clonogenic potential as compared to the uncultured cells. Four weeks after transplantation, 73% (19/26) of expanded-cell recipients and 35% (7/20) of unexpanded-cell recipients were found to be successfully engrafted with human CD45+ cells. The engraftment level of expanded MNCs was significantly (1.8-fold) higher than unexpanded cells. After hHGFs administration, the level was increased to 3.2, 3.8 and 2.6-fold at respectively 8, 12, and 16 weeks of post transplantation. The increased expression of CXCR4 protein in expanded MNCs is a likely explanation for the present findings. CONCLUSION: The presented data showed that expanded MNCs compared to unexpended cells are capable of more rapid and higher short-term engraftment in normal fetal mouse. It could also be suggested that in utero transplantation (IUT) of normal fetal mice could be an appropriate substitute for NOD/SCID mice in xenotransplantation studies.

Umbilical cord blood quality and quantity: Collection up to transplantation.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells for transplantation in some blood disorders. One of the major factors that influence on transplantation fate is cord blood (CB) cell count, in addition to human leukocyte antigen similarity and CD34+ cell number. Here, we review the factors that could effect on quality and quantity of CBUs. Relevant English-language literatures were searched and retrieved from PubMed using the terms: CB, quality, collection, and transplantation. The numbers of total nucleated cells (TNCs) and CD34+ cells are good indicators of CB quality because they have been associated with engraftment; thereby, whatever the TNCs in a CB unit (CBU) are higher, more likely they led to successful engraftment. Many factors influence the quantity and quality of UCB units that collect after delivery. Some parameters are not in our hands, such as maternal and infant factors, and hence, we cannot change these. However, some other factors are in our authority, such as mode of collection, type and amount of anticoagulant, and time and temperature during collection to postthaw CBUs and freeze-and-thaw procedures. By optimizing the CB collection, we can improve the quantity and quality of UCB for storage and increase the likelihood of its use for transplantation.

Manipulation of Hematopoietic Stem Cell Fate by Small Molecule Compounds.

Self-renewal and multipotential differentiation are two important features of hematopoietic stem/progenitor cells (HS/PCs) that make them as an ideal source of stem cells for treatment of many hematologic disorders and cancers. Regarding the limited number of cord blood HS/PCs, proper ex vivo expansion can significantly increase the clinical use of cord blood stem cells. Meanwhile, expansion of HS/PCs will be feasible through bypassing the quiescent state of HS/PCs and simultaneously enhancing their proliferative potential and survival while delaying the terminal differentiation and exhaustion. Previous investigations have demonstrated that defined sets of exogenous hematopoietic cytokines/growth factors such as stem cell factor, Flt-3 ligand, and thrombopoietin are able to expand HS/PCs. However, in recent years, small molecule compounds (SMCs) have emerged as a powerful tool for the effective expansion of HS/PCs by modulating multiple cellular processes including different signaling pathways and epigenetics. In this review, recent progress toward the use of SMCs in HS cell research is presented. We focus on the significant applications of SMCs related to HS/PC expansion and discuss the associated mechanism. At the end we present a list of those SMCs which enter to clinical trials.