Jelly-derived Mesenchymal Stromal Cells Research Articles

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

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

Human Wharton's jelly-derived mesenchymal stromal stem cells preconditioned with valproic acid promote cell migration and reduce renal inflammation in ischemia/reperfusion injury by activating the AKT/P13K and SDF1/CXCR4 pathways

ObjectiveTo determine whether WJ-MSCs pretreated with VPA would enhance their migration to improve functional recovery of renal IRI in rats. Methods150 Sprague-Dawley rats were distributed into 5 groups; Sham, IRI, WJ-MSC, VPA, and WJ-MSCs + VPA. 10 rats were sacrificed after 3, 5, and 7 days. Role of WJ-MSCs pretreated with VPA was evaluated by assessment of renal function, antioxidant enzymes together with renal histopathological and immunohistopathological analyses and finally by molecular studies. ResultsWJ-MSCs and VPA significantly improved renal function and increased antioxidants compared to IRI group. Regarding gene expression, WJ-MSCs and VPA decreased BAX and TGF-β1, up-regulated Akt, PI3K, BCL2, SDF1α, and CXCR4 related to IRI. Additionally, WJ-MSCs pretreated with VPA improved the measured parameters more than either treatment alone. ConclusionWJ-MSCs isolated from the umbilical cord and pretreated with VPA defended the kidney against IRI by more easily homing to the site of injury.

Mesenchymal Stromal Cells from Human Wharton's Jelly Modulate the Intraocular Immune Response in a Glucocorticoid Hypertension Model: An Exploratory Analysis.

Glaucoma is a neurodegenerative disease characterized by the loss of retinal ganglion cells. Recent research suggests immunological changes such as cytokine imbalance may affect its pathophysiology. This implies that immunomodulation, like that of mesenchymal cells, could be a potential therapeutic avenue for this disease. However, the effects of intravitreal injections of human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) on intraocular immune response have not been assessed in ocular hypertension (OH) models. We explored this by measuring cytokine levels and expression of other markers, such as glial fibrillary acidic protein (GFAP) and T cells, in 15 randomly divided New Zealand rabbits: G1: OH, G2: hWJ-MSCs, and G3: OH+hWJ-MSCs. We analyzed the aqueous humor (IL-6, IL-8, and TNF-α) and vitreous humor (IFN-γ, IL-10, and TGF-β) using ELISA and flow cytometry (cell populations), as well as TCD3+, TCD3+/TCD4+, and TCD3+/TCD8+ lymphocytes, and GFAP in the retina and optic nerve through immunohistochemistry. We found a decrease in TNF-α, IL-6, IFN-γ, IL-10, and IL-8 in G3 compared to G1 and an increase in TGF-β in both G2 and G3. TCD3+ retinal infiltration in all groups was primarily TCD8+ rather than TCD4+ cells, and strong GFAP expression was observed in both the retina and optic nerves in all groups. Our results suggest that cellular and humoral immune responses may play a role in glaucomatous optic neuropathy and that intravitreal hWJ-MSCs can induce an immunosuppressive environment by inhibiting proinflammatory cytokines and enhancing regulatory cytokines.

Early detection of optic nerve head changes using optical coherence tomography after using mesenchymal stromal cells as intravitreal therapy in rabbit models of ocular hypertension.

Stem cell therapy is considered a promising treatment for several neurodegenerative diseases. However, there are very few studies on the use of this therapy in glaucoma models. By detecting the changes produced by glaucoma early, cell therapy could help prevent the events that lead to blindness. In this study, early changes in the optic nerve head (ONH) as detected by optical coherence tomography (OCT) after the application of human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) in an experimental model of ocular hypertension (OH) were evaluated. Fifteen New Zealand rabbits were randomly divided into the following three groups: G1: OH, G2: hWJ-MSCs, and G3: OH + hWJ-MSCs. An OH model was constructed, and the intraocular pressure (IOP) was measured regularly. At week 7, 105/100 μL hWJ-MSCs were intravitreally injected. Retinography and OCT were used to evaluate structural changes in ONH. IOP increased significantly in G1 and G3 from week 3 onward. Retinography revealed more significant optic nerve changes, that is, papillary asymmetry suggestive of optic nerve excavation, vascular alterations, and irregular hypopigmentation peripheral to the optic disk margin, in G1 compared with G3. OH locates the hWJ-MSCs solution in the vitreous in front of the optic nerve. OCT revealed retinal nerve fiber layer (RNFL) reduction in all groups, reduced optic cup volume in G2 and G3 between weeks 1 and 9, and significant ganglion cell layer thickness reduction in G1 and a slight increase in G3. Intravitreal hWJ-MSCs injection produced changes in optic cup volume, which were detected early on by OCT; however, RNFL could not be restored in this OH model.

Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors.

Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47×109 cells in a real-scale 2.4-L batch.

Efficacy of Wharton Jelly Mesenchymal Stromal Cells infusions in moderate to severe SARS-Cov-2 related acute respiratory distress syndrome: a phase 2a double-blind randomized controlled trial.

The COVID-19 pandemic caused a wave of acute respiratory distress syndrome (ARDS) with a high in-hospital mortality, especially in patients requiring invasive mechanical ventilation. Wharton Jelly-derived Mesenchymal Stromal Cells (WJ-MSCs) may counteract the pulmonary damage induced by the SARS-CoV-2 infection through pro-angiogenic effects, lung epithelial cell protection, and immunomodulation. In this randomized, double-blind, placebo-controlled phase 2a trial, adult patients receiving invasive mechanical ventilation for SARS-CoV-2 induced moderate or severe ARDS were assigned to receive 1 intravenous infusion of 1 × 106 WJ-MSCs/kg or placebo within 48 h of invasive ventilation followed by 2 infusions of 0.5 × 106 WJ-MSCs/kg or placebo over 5 days. The primary endpoint was the percentage of patients with a PaO2/FiO2 > 200 on day 10. Thirty patients were included from November 2020 to May 2021, 15 in the WJ-MSC group and 15 in the placebo group. We did not find any significant difference in the PaO2/FiO2 ratio at day 10, with 18 and 15% of WJ-MSCs and placebo-treated patients reaching a ratio >200, respectively. Survival did not differ in the 2 groups with a 20% mortality rate at day 90. While we observed a higher number of ventilation-free days at 28 days in the WJ-MSC arm, this difference was not statistically significant (median of 11 (0-22) vs. 0 (0-18), p = 0.2). The infusions were well tolerated, with a low incidence of anti-HLA alloimmunization after 90 days. While treatment with WJ-MSCs appeared safe and feasible in patients with SARS-CoV2 moderate or severe ARDS in this phase 2a trial, the treatment was not associated with an increased percentage of patients with P/F > 200 at 10d, nor did 90 day mortality improve in the treated group. https://beta.clinicaltrials.gov/study/NCT04625738, identifier NCT04625738.

The effect of mesenchymal stromal cells of different origin on morphological parameters in the somatosensory cortex of rats with acute cerebral ischemia

Ischemic stroke is the second leading cause of death and the leading cause of disability worldwide. So far, promising experimental data have been obtained regarding the elimination of neurological dysfunction and the reduction of the area of ischemic damage when using mesenchymal stromal cells (MSCs). Purpose: to characterize the influence of MSCs of different origin, MSC lysate of human Wharton cells and citicoline on the dynamics of destructive changes in the somatosensory cortex of rats with acute cerebrovascular accident according to light microscopy and micromorphometry data. Materials and methods. An experiment was performed using 190 -4-month-old male Wistar rats weighing 160-190 g, which were subjected to transient bilateral 20-minute ischemia-reperfusion (IR) of the internal carotid arteries. After modeling the pathology, the animals were injected into the femoral vein with obtained from human umbilical cord Wharton’s jelly-derived MSCs, human and rat adipose tissue-derived MSCs at a dose of 106 cells/animal. Other groups of experimental animals were intravenously injected with fetal rat fibroblasts at a dose of 106 cells/animal in 0.2 ml of physiological solution and lysate of human umbilical cord Wharton’s jelly-derived MSCs at a dose of 0.2 ml/animal. Control animals were injected IV with 0.2 ml of physiological solution. The last group of rats received a single dose of the reference drug citicoline at a dose of 250 mg/kg. The studies were conducted on the 7th and 14th day. In the somatosensory cortex, the total number of neuron nuclei per 1 mm2 was counted, and the ratio of the number of intact neuron nuclei and nuclei with pathological changes (karyorrhexis and karyopyknosis) was also determined. Results: The transplantation of stem cells, lysate of human umbilical cord Wharton’s jelly-derived MSCs, or citicoline contributed to an increase in the number of neurons with nuclei in the somatosensory cortex, as well as an increase in the number of nuclei that did not undergo pathological changes. The transplantation of human umbilical cord Wharton’s jelly-derived MSCs had the most positive effect. The number of neuron nuclei in 1 mm2 that did not undergo pathological changes in the somatosensory cortex in this group of animals approached the number of nuclei in the group of pseudo-operated animals, while the number of nuclei that did not undergo pathological changes significantly exceeded the number of nuclei with signs of destruction. Conclusion: A significant increase in the number of neurons without signs of pathological changes was observed in all experimental groups of rats during the simulation of ischemic brain damage after the introduction of various types of studied mesenchymal stromal cells, lysate or citicoline. The most positive result in the somatosensory cortex was achieved after the introduction of human umbilical cord Wharton’s jelly-derived MSCs.

Umbilical cord-derived mesenchymal stromal cells preserve endogenous insulin production in type 1 diabetes: a Phase I/II randomised double-blind placebo-controlled trial

Aim/hypothesisThis study aimed to investigate the safety and efficacy of treatment with allogeneic Wharton’s jelly-derived mesenchymal stromal cells (MSCs) in recent-onset type 1 diabetes.MethodsA combined Phase I/II trial, composed of a dose escalation followed by a randomised double-blind placebo-controlled study in parallel design, was performed in which treatment with allogeneic MSCs produced as an advanced therapy medicinal product (ProTrans) was compared with placebo in adults with newly diagnosed type 1 diabetes. Inclusion criteria were a diagnosis of type 1 diabetes <2 years before enrolment, age 18–40 years and a fasting plasma C-peptide concentration >0.12 nmol/l. Randomisation was performed with a web-based randomisation system, with a randomisation code created prior to the start of the study. The randomisation was made in blocks, with participants randomised to ProTrans or placebo treatment. Randomisation envelopes were kept at the clinic in a locked room, with study staff opening the envelopes at the baseline visits. All participants and study personnel were blinded to group assignment. The study was conducted at Karolinska University Hospital, Stockholm, Sweden.ResultsThree participants were included in each dose cohort during the first part of the study. Fifteen participants were randomised in the second part of the study, with ten participants assigned to ProTrans treatment and five to placebo. All participants were analysed for the primary and secondary outcomes. No serious adverse events related to treatment were observed and, overall, few adverse events (mainly mild upper respiratory tract infections) were reported in the active treatment and placebo arms. The primary efficacy endpoint was defined as Δ-change in C-peptide AUC for a mixed meal tolerance test at 1 year following ProTrans/placebo infusion compared with baseline performance prior to treatment. C-peptide levels in placebo-treated individuals declined by 47%, whereas those in ProTrans-treated individuals declined by only 10% (p<0.05). Similarly, insulin requirements increased in placebo-treated individuals by a median of 10 U/day, whereas insulin needs of ProTrans-treated individuals did not change over the follow-up period of 12 months (p<0.05).Conclusions/interpretationThis study suggests that allogeneic Wharton’s jelly-derived MSCs (ProTrans) is a safe treatment for recent-onset type 1 diabetes, with the potential to preserve beta cell function.Trial registrationClinicalTrials.gov NCT03406585FundingThe sponsor of the clinical trial is NextCell Pharma AB, Stockholm, Sweden.Graphical

The effect of mesenchymal stromal cells of various origins on morphology of hippocampal CA1 area of rats with acute cerebral ischemia

Every year, about 150,000 strokes occur in Ukraine, and more than 100,000 people die from the consequences of stroke and other circulatory disorders in the brain. So far, promising experimental data on the treatment of neurological dysfunction using mesenchymal stromal cells (MSCs) have been obtained. Purpose: to characterize the impact of MSCs of various origins, lysate of Wharton’s jelly-derived MSCs and citicoline on the dynamics of destructive changes in the hippocampal CA1 area of rats with model of acute cerebral ischemia according to morphometric data. Materials and methods. An experiment was performed using 4-month-old male Wistar rats, which were subjected to transient bilateral 20-minute ischemia-reperfusion (IR) of the internal carotid arteries. After modeling, the animals were injected intravenously with Wharton’s jelly-derived MSCs, human and rat adipose-derived MSCs at a dose 106 cells/animal. Other groups were intravenously injected with rat fetal fibroblasts at a dose of 106 cells/animal and lysate from Wharton’s umbilical cord MSCs at a dose of 0.2 mL/animal. Control animals were injected with 0.2 mL of saline. The last group of rats received a single dose of the reference drug citicoline at a dose of 250 mg/kg. On the 7th and 14th day, the total number of neuron nuclei per 1 mm2 brain section was counted in the hippocampal CA1 area, and the ratio of the number of intact neuron nuclei and nuclei with changes (karyorrhexis and karyopyknosis) was determined. Results. The transplantation of MSCs, lysate of Wharton’s jelly-derived MSCs, or citicoline contributed to a greater value of the number of nuclei in the hippocampal CA1 area, and the number of nuclei that did not undergo pathological changes also increased. The transplantation of Wharton’s jelly-derived MSCs had the most positive effect. The number of neuron nuclei per 1 mm2 in the hippocampal CA1 area in this group of animals approached the number of nuclei in the group of sham-operated animals. At the same time, the number of nuclei that did not undergo pathological changes significantly exceeded the number of nuclei with signs of destruction. Conclusion. A significant increase in the number of neurons without signs of pathological changes was observed in all experimental groups of rats during the modeling of ischemic brain injury after the administration of various types of studied mesenchymal stromal cells, lysate or citicoline. The most positive result in the hippocampal CA1 area was achieved after the administration of Wharton’s jelly-derived MSCs.

Long-term passages of human clonal mesenchymal stromal cells can alleviate the disease in the rat model of collagen-induced arthritis resembling early passages of different heterogeneous cells.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown cause. The interaction of immune system cells and the secretion of inflammatory cytokines with synovial cells leads to severe inflammation in the affected joints. Currently, medications, including non-steroidal anti-inflammatory drugs, glucocorticoids, and more recently, disease-modifying anti-rheumatic drugs, are used to reduce inflammation. However, long-term use of these drugs causes adverse effects or resistance in a considerable number of RA patients. Recent findings revealed the safety and efficacy of mesenchymal stromal cells (MSCs)-based therapies both in RA animal models and clinical trials. Here, the beneficial effects of bone marrow-derived heterogeneous MSCs (BM-hMSCs) and Wharton jelly-derived MSCs (WJ-MSCs) at early passages were compared to BM-derived clonal MSCs (BM-cMSCs) at high passage number on a rat model of collagen-induced arthritis. Results showed that systemic delivery of MSCs significantly reversed adverse changes in body weight, paw swelling, and arthritis score in all MSC-treated groups. Radiological images and histological evaluation demonstrated the therapeutic effects of MSCs. There was a decrease in serum level of anti-collagen type II immunoglobulin G and the inflammatory cytokines interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor-α in all MSC-treated groups. In contrast, an increase in inhibitory cytokines transforming growth factor-β and IL-10 was seen. Notably, the long-term passages of BM-cMSCs could alleviate RA symptoms similar to the early passages of WJ-MSCs and BM-hMSCs. The importance of BM-cMSCs is the potential to establish cell banks with billions of cells derived from a single donor that could be a competitive cell-based therapy to treat RA.

Assessment of clinical, biochemical, and radiological outcomes following intra-articular injection of Wharton jelly-derived mesenchymal stromal cells in patients with knee osteoarthritis: A prospective clinical study.

The aim of the present study was to perform clinical, biochemical, and radiological evaluation of the efficacy of mesenchymal stem cells derived from Wharton jelly (WJ) present within the human umbilical cord in the treatment of knee osteoarthritis. Between 2018 and 2019, 10 patients with knee osteoarthritis for whom the conservative treatment was not beneficial were included in the study. Patients were clinically, radiologically, and biochemically evaluated before treatment initiation. Thereafter, the patients were intra-articularly injected using a solution containing 1 × 108 WJ-derived MSCs. Evaluations were performed on day 21 (V1) and 42 (V2) and month 3 (V3), 6 (V4), and 12 (V5) after the procedure. At 1-year post-injection, visual analogue scale, Western Ontario and McMaster Universities Osteoarthritis Index, and Lequesne scores of patients were lower than those observed during the initial evaluation, whereas the mean 36-Item Short Form Health Survey score was higher. Cartilage thicknesses were found to be increased in all regions except in the medial femur, medial posterior femur, lateral posterior femur, and lateral posterior tibia regions in magnetic resonance imaging. A significant increase was observed in tumor necrosis factor-alpha, interleukin-1β, adiponectin, resistin, and interleukin-6 levels compared with pre-injection values. The leptin levels at 6-month and 1-year controls were lower than the pre-injection levels, and the decrease observed at 6 months was significant. In patients with knee osteoarthritis, intra-articular WJ-derived MSC injection causes significant pain reduction, satisfactory functional improvement, and increased patient satisfaction following a 1-year follow-up. These clinical improvements were supported by magnetic resonance images, along with changes in adiponectin and leptin levels in synovial fluid.Level of evidence: IV.

Shipping Temperature, Time and Media Effects on Equine Wharton's Jelly and Adipose Tissue Derived Mesenchymal Stromal Cells Characteristics.

Simple SummaryToday, the use of horse adipose tissue and Wharton’s jelly-derived mesenchymal stromal cells in veterinary regenerative medicine represents a promising tool. Cells need to be isolated and expanded in vitro in the laboratory to obtain a sufficient amount for clinical application and its characterization. In many cases, laboratories and clinics where the therapy will be performed are in different and far-flung facilities, and the cells must therefore be shipped by a courier. The authors evaluated the effects of different storage conditions, in terms of temperature, time of storage and storage solutions on cell viability, cell growth, differentiation potential and molecular characteristics. The aim was to state the most appropriate storage conditions for transporting adipose tissue and Wharton’s jelly-derived stromal cells, ensuring the maintenance of the stemness features for therapeutic application in horses. To use Mesenchymal Stromal Cells (MSCs) in equine patients, isolation and expansion are performed in a laboratory. Cells are then sent back to the veterinary clinic. The main goal of storage conditions during cell transport is to preserve their biological properties and viability. The aim of this study was to evaluate the effects of storage solutions, temperature and time on the characteristics of equine adipose tissue and Wharton’s jelly-derived MSCs. We compared two different storage solutions (plasma and 0.9% NaCl), two different temperatures (4 °C and room temperature) and three time frames (6, 24, 48 h). Cell viability, colony-forming units, trilineage differentiation, the expression of CD45 and CD90 antigens and adhesion potentials were evaluated. Despite the molecular characterization and differentiation potential were not influenced by storage conditions, viability, colony-forming units and adhesion potential are influenced in different way, depending on MSCs sources. Overall, this study found that, despite equine adipose tissue MSCs being usable after 24 h of storage, cells derived from Wharton’s jelly need to be used within 6 h. Moreover, while for adipose cells the best conservation solutions seems to be plasma, the cell viability of Wharton’s jelly MSCs declined in both saline and plasma solution, confirming their reduced resistance to conservation.

The effect of mesenchymal stromal cells of various origins on mortality and neurologic deficit in acute ischemia-reperfusion in rats

Stroke is a global epidemic issue and the second leading cause of death in the world and in Ukraine. According to official statistics, every year 100-110 thousand Ukrainians suffer acute cerebrovascular disorders. One third of such patients are of working age, up to 50 % will have a disability, and only one in ten will fully return to full life. So far, promising experimental data on the treatment of neurological dysfunction using mesenchymal stromal cells (MSCs) have been obtained. The aim of study is to compare the effect of MSCs of different origins on mortality and neurologic deficit in rats with acute cerebral ischemia-reperfusion injury (CIRI). Materials and methods. Transient bilateral 20-minute occlusion of internal carotid arteries was modeled in male Wistar rats aged 4 months and animals were injected intravenously with MSCs derived from human umbilical cord Wharton's-jelly (hWJ-MSC), human and rat adipose tissue. Other groups of experimental animals were injected intravenously with rat fetal fibroblasts and cell lysate from hWJ-MSC. The last group of rats received Citicoline at a dose of 250 mg/kg as a reference drug. Control animals were injected intravenously with normal saline. The cerebroprotective effect of therapy was assessed by mortality and neurologic deficit in rats on the McGraw's stroke index score. Results. After 12 hours of observation in the crucial period in the development of experimental acute cerebrovascular disorders with the administration of hWJ-MSC, mortality was only 10 % against 45 % of animals in the control group. The use of rat fetal fibroblasts reduced the mortality of animals compare to the control group by an average of 25 %. CIRI in rats caused severe neurologic deficits: paralysis, paresis, ptosis, circling behavior. On the 7th day of observation in the control group of animals, the mean score on the McGrow's stroke index indicated severe neurological disorders. On the 14th day of observation in this group of animals there was no complete recovery of lost central nervous system functions. Compared with the control group of animals, all the treatment agents for acute CIRI (MSCs of various origins, MSC's lysate and Citicoline) contributed to a significant regression of neurologic deficit. Conclusions. Thus, transplantation of human Wharton's jelly-derived MSCs and rat fetal fibroblasts reduced mortality and alleviated neurological symptoms in rats with experimental ischemic stroke. hWJ-MSC, rat fetal fibroblasts, and rat adipose-derived MSCs reduced the incidence of neurological disorders better than Citicoline, which was accompanied by a regression of neurologic deficit dynamics on the 14th day of follow-up. The ability of stem cells of different origins to reduce neurologic deficit indicates the feasibility of their use in experimental acute cerebral ischemia.

Human Wharton's Jelly-Derived Mesenchymal Stromal Cells Primed by Tumor Necrosis Factor-α and Interferon-γ Modulate the Innate and Adaptive Immune Cells of Type 1 Diabetic Patients.

The unique immunomodulation and immunosuppressive potential of Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) make them a promising therapeutic approach for autoimmune diseases including type 1 diabetes (T1D). The immunomodulatory effect of MSCs is exerted either by cell-cell contact or by secretome secretion. Cell-cell contact is a critical mechanism by which MSCs regulate immune-responses and generate immune regulatory cells such as tolerogenic dendritic cells (tolDCs) and regulatory T cell (Tregs). In this study, we primed WJ-MSCs with TNF-α and IFN-γ and investigated the immunomodulatory properties of primed WJ-MSCs on mature dendritic cells (mDCs) and activated T cells differentiated from mononuclear cells (MNCs) of T1D patient’s. Our findings revealed that primed WJ-MSCs impaired the antigen-mediated immunity, upregulated immune-tolerance genes and downregulated immune-response genes. We also found an increase in the production of anti-inflammatory cytokines and suppression of the production of pro-inflammatory cytokines. Significant upregulation of FOXP3, IL10 and TGFB1 augmented an immunosuppressive effect on adaptive T cell immunity which represented a strong evidence in support of the formation of Tregs. Furthermore, upregulation of many critical genes involved in the immune-tolerance mechanism (IDO1 and PTGES2/PTGS) was detected. Interestingly, upregulation of ENTPD1/NT5E genes express a strong evidence to switch immunostimulatory response toward immunoregulatory response. We conclude that WJ-MSCs primed by TNF-α and IFN-γ may represent a promising tool to treat the autoimmune disorders and can provide a new evidence to consider MSCs- based therapeutic approach for the treatment of TID.

Evaluation of Potential Application of Wharton’s Jelly-Derived Human Mesenchymal Stromal Cells and its Conditioned Media for Dermal Regeneration using Rat Wound Healing Model

Mesenchymal stromal cells and the derived conditioned media represent an area of tremendous medical interest and, among other clinical applications, are currently being extensively explored for wound healing. The aim of this study was to comparatively evaluate the wound healing potential of xeno-free human umbilical cord-derived mesenchymal stromal cells (MSCs) and the conditioned media (CM) in a full-thickness excision wound model in rats. The evaluation parameters included rate of wound healing, serum cytokine analyses, collagen content, histopathology, and hyperspectral imaging as an independent qualitative and quantitative tool. Both the cell-based and cell-free approaches scored better in lower inflammation, as evidenced in lower IL-10 and stable IL-6 levels, and improved rate of wound healing (p < 0.0001). More importantly, no adverse reaction or rejection was observed although human MSCs and CM were used in a xenogeneic model. The presence of hFGF, hHGF, hGCSF, hIL-1Ra, hVEGF, and hIL-6 in the secretome may elucidate the regenerative potential of the xeno-free cell-based and cell-free approaches which have translational value for advanced wound care. The results revealed the therapeutic potential of both the cell-based and cell-free approaches for wound healing.

Therapeutic effects of Wharton’s jelly-derived Mesenchymal Stromal Cells on behaviors, EEG changes and IGF-1 in rat model of the Parkinson’s disease

Human Wharton's jelly-derived Mesenchymal Stromal Cells (hWJ-MSCs) have shown beneficial effects in improving the dopaminergic cells in the Parkinson’s disease (PD). In the present study, the effects of hWJ-MSCs on hyperalgesia, anxiety deficiency and Pallidal local electroencephalogram (EEG) impairment, alone and combined with L-dopa, were examined in a rat model of PD.Adult male Wistar rats were divided into five groups: 1) sham, 2) PD, 3) PD + C (Cell therapy), 4) PD + C+D (Drug), and 5) PD + D. PD was induced by injection of 6-OHDA (16 μg/2 μl into medial forebrain bundle (MFB)). PD + C group received hWJ-MSCs (1 × 106 cells, intravenous (i.v.)) twice post PD induction. PD + C+D groups received hWJ-MSCs combined with L-Dopa/Carbidopa, (10/30 mg/kg, intraperitoneally (i.p.)). PD + D group received L-Dopa/Carbidopa alone. Four months later, analgesia, anxiety-like behaviors, were evaluated and Pallidal local EEG was recorded. Level of insulin-like growth factor 1 (IGF-1) was measured in the striatum and dopaminergic neurons were counted in substantia nigra (SNc).According to data, MFB-lesioned rats showed hyperalgesia in tail flick, anxiety-like symptoms in cognitive tests, impairment of electrical power of pallidal local EEG as field potential, count of dopaminergic neurons in SNc and level of IGF-1 in striatum. These complications restored significantly by MSCs treatment (p < 0.001).Our findings confirm that chronic treatment with hWJ-MSC, alone and in combination with L-Dopa, improved nociception and cognitive deficit in PD rats which may be the result of increasing IGF-1 and protect the viability of dopaminergic neurons.

Derivation of Mesenchymal Stromal Cells from Ovine Umbilical Cord Wharton's Jelly.

The methods described herein allow for the isolation and expansion of fibroblastic-like ovine Wharton's jelly-derived mesenchymal stromal cells (oWJ-MSC) that, similarly to their human counterparts, adhere to standard plastic surfaces in culture; show a mesenchymal profile for specific surface antigens (i.e., positive for CD44 and CD166); and lack expression of endothelial (CD31) and hematopoietic (CD45) markers as well as major histocompatibility complex (MHC) class-II. Homogeneous cell cultures result from a two-phase bioprocess design that starts with the isolation of mesenchymal stromal cells (MSC) from the Wharton's jelly of ovine umbilical cords up to a first step of cryopreservation. The second phase allows for further expansion of ovine WJ-MSC up to sufficient numbers for further studies. Overall, this methodology encompasses a 2-week bioprocess design that encompasses two cell culture passages ensuring sufficient cells for the generation of a Master Cell Bank. Further thawing and scale expansion results in large quantities of oWJ-MSC that can be readily used in proof of efficacy and safety studies in the preclinical development stage of the development of cell-based medicines. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation and expansion of ovine mesenchymal stromal cells from Wharton's jelly of the umbilical cord Basic Protocol 2: Characterization of ovine mesenchymal stromal cells Basic Protocol 3: Growth profile determination of ovine mesenchymal stromal cells from Wharton's jelly.

A Xeno-Free Strategy for Derivation of Human Umbilical Vein Endothelial Cells and Wharton's Jelly Derived Mesenchymal Stromal Cells: A Feasibility Study toward Personal Cell and Vascular Based Therapy.

Coimplantation of endothelial cells (ECs) and mesenchymal stromal cells (MSCs) into the transplantation site could be a feasible option to achieve a sufficient level of graft-host vascularization. To find a suitable source of tissue that provides a large number of high-quality ECs and MSCs suited for future clinical application, we developed a simplified xeno-free strategy for isolation of human umbilical vein endothelial cells (HUVECs) and Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from the same umbilical cord. We also assessed whether the coculture of HUVECs and WJ-MSCs derived from the same umbilical cord (autogenic cell source) or from different umbilical cords (allogenic cell sources) had an impact on in vitro angiogenic capacity. We found that HUVECs grown in 5 ng/ml epidermal growth factor (EGF) supplemented xeno-free condition showed higher proliferation potential compared to other conditions. HUVECs and WJ-MSCs obtained from this technic show an endothelial lineage (CD31 and von Willebrand factor) and MSC (CD73, CD90, and CD105) immunophenotype characteristic with high purity, respectively. It was also found that only the coculture of HUVEC/WJ-MSC, but not HUVEC or WJ-MSC mono-culture, provides a positive effect on vessel-like structure (VLS) formation, in vitro. Further investigations are needed to clarify the pros and cons of using autogenic or allogenic source of EC/MSC in tissue engineering applications. To the best of our knowledge, this study offers a simple, but reliable, xeno-free strategy to establish ECs and MSCs from the same umbilical cord, a new opportunity to facilitate the development of personal cell-based therapy.

TWIST1-Reprogrammed Endothelial Cell Transplantation Potentiates Neovascularization-Mediated Diabetic Wound Tissue Regeneration.

Hypovascularized diabetic nonhealing wounds are due to reduced number and impaired physiology of endogenous endothelial progenitor cell (EPC) population that limits their recruitment and mobilization at the wound site. For enrichment of the EPC repertoire from nonendothelial precursors, abundantly available mesenchymal stromal cells (MSC) were reprogrammed into induced endothelial cells (iEC). We identified cell signaling molecular targets by meta-analysis of microarray data sets. BMP-2 induction leads to the expression of inhibitory Smad 6/7-dependent negative transcriptional regulation of ID1, rendering the latter's reduced binding to TWIST1 during transdifferentiation of Wharton jelly-derived MSC (WJ-MSC) into iEC. TWIST1, in turn, regulates endothelial gene transcription, positively of proangiogenic KDR and negatively, in part, of antiangiogenic SFRP4 Twist1 reprogramming enhanced the endothelial lineage commitment of WJ-MSC and increased the vasculogenic potential of reprogrammed endothelial cells (rEC). Transplantation of stable TWIST1 rEC into a type 1 and 2 diabetic full-thickness splinted wound healing murine model enhanced the microcirculatory blood flow and accelerated the wound tissue regeneration. An increased or decreased colocalization of GFP with KDR/SFRP4 and CD31 in the regenerated diabetic wound bed with TWIST1 overexpression or silencing (piLenti-TWIST1-shRNA-GFP), respectively, further confirmed improved neovascularization. This study depicted the reprogramming of WJ-MSC into rEC using unique transcription factor TWIST1 for an efficacious cell transplantation therapy to induce neovascularization-mediated diabetic wound tissue regeneration.

Xenogeneic transplantation of human WJ-MSCs rescues mice from acute radiation syndrome via Nrf-2-dependent regeneration of damaged tissues.

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ-MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue-regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ-MSCs protected mice from a lethal dose of IR even when transplanted up to 24hours after irradiation, and a combination of WJ-MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ-MSCs. This combinatorial approach mitigated IR-induced damage to the hematopoietic and gastrointestinal system. WJ-MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony-stimulating factor (G-CSF) and IL-6 in mice. Knockdown of G-CSF and IL-6 in WJ-MSCs before injection to lethally irradiated mice or transplantation of WJ-MSCs to lethally irradiated Nrf-2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ-MSCs could be a potential cell-based therapy for individuals accidentally exposed to radiation.