Effects of Mesenchymal Stem Cell-conditioned Media with Natural Immunomodulatory Agent Resveratrol on Type 1 Diabetes.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a condition marked by elevated blood sugar levels and primarily recognized by the destruction of beta cells caused by an autoimmune attack, which is a significant characteristic of T1DM. Recent studies have demonstrated the regenerative potential of conditioned medium therapy. In light of this, the current research sought to assess the impact of Mesenchymal Stem Cell conditioned media (CM) and CM with resveratrol (CM+ Resveratrol) on the management of T1DM in Swiss albino mice. By leveraging and modifying existing conditioned medium therapy, this study aims to evaluate its effectiveness in treating T1DM. MATERIALS & METHODS: Diabetes was induced in animals using the diabetes-inducing agent streptozotocin (STZ). The animals were then divided into five groups: Normal control, Disease Control, Resveratrol, Condition Media, and CM + Resveratrol. Treatments were given to the animals accordingly. The study period was 28 days. During this time, the animals were monitored for foodwater intake twice a week, blood glucose levels, and body weight. At the conclusion of the 28-day study period, biochemical estimations were performed for serum insulin levels, C-peptide levels, anti-inflammatory cytokines levels and pro-inflammatory cytokines levels. Additionally, histopathology of the pancreas was performed. RESULTS: The test groups showed a significant decrease in blood glucose levels, an increase in Cpeptide levels, and a decrease in pro-inflammatory cytokine levels compared to the disease group. However, no statistically significant change within groups was observed in terms of serum insulin and anti-inflammatory cytokine levels. The improvement in diabetic symptoms, such as polyphagia, polydipsia, and weight loss, was observed in the treatment group, along with pancreatic regeneration, which indicated improved insulin secretion. CONCLUSION: In the current investigation, we concluded that CM and CM+ Resveratrol, as natural immunomodulators, have the capacity to regenerate injured pancreatic beta cells and have antidiabetic action, together with immunomodulating impact. Nonetheless, future studies on this therapy appear to be promising.

Long-term cryopreservation of whole gingival tissue.

Stem cells obtained from the body tissue, such as adipose tissue, dental pulp and gingival tissue. Fresh tissue is often used to isolate and culture for regenerative medicine. However, availability of tissue as and when required is one of the measure issue in regenerative medicine. Cryopreservation of tissue provides benefit over tissue availability, storage for significant amount of period and helps preserve the original cell structures. The effects of cryopreservation of gingival tissue for mesenchymal stem cell (MSC) are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation on the long term survival the whole gingival biopsy tissue. We studied cell outgrowth, cell morphology, MSC surface-markers and differentiation of mesenchymal stem cells derived from cryopreserved gingiva. In this study, gingival tissue was cryopreserved for 3, 6, 9 months. Cryopreserved tissue has been thawed and cells were isolated by using explant culture method. The fresh and cryopreserved gingival tissue cells were cultured and characterized for surface marker analysis, CFU-f, population doubling time, and osteogenic, chondrogenic and adipogenic differentiation. The fresh and cryopreserved tissue has similar stem cell properties. Results indicate that cryopreservation of the entire gingival tissue does not affect the properties of stem cells. This opens door for gingival tissue banking for future use in periodontology and regenerative medicine.

Ascorbic acid and IFNγ preconditioning enhance the potency of human mesenchymal stem cells to ameliorate LPS induced cytokine storm.

The mesenchymal Stem Cells (MSCs) is one of the leading contender in therapeutic management of cytokine storm implicated in the COVID-19 and other inflammatory conditions. This study was aimed to investigate the effect of Interferon gamma (IFN-γ) and Ascorbic Acid (AA) preconditioning on the secretome of the human Umbilical Cord Derived MSCs (UCMSCs) and their potential to ameliorate the lipopolysaccharide (LPS) induced cytokine storm in the human peripheral blood mononuclear cells (PBMCs). UCMSCs were preconditioned with IFN-γ, AA and secretome (UCMSCs-S, IFNγ-UCMSCs-S and AA-UCSMCs-S) was analysed for the levels of growth factors and cytokines by flow cytometry. The potential of secretome to ameliorate cytokine storm and augment angiogenesis was assessed in the LPS induced PBMCs and yolk sac membrane (YSM) assay respectively. The mRNA transcript and protein levels of IL-6, IL-1ß and TNF-α was analysed by RT-PCR and flow cytometry respectively. IFNγ-UCMSCs-S and AA-UCSMCs-S ameliorated the LPS induced cytokine storm as revealed by the decreased mRNA and protein expression of IL-6, IL-1ß and TNF-α as compared to the UCMSCs-S. IFNγ-UCMSCs-S and AA-UCSMCs-S augmented angiogenesis in YSM assay. Furthermore, IFNγ and AA preconditioning of UCMSCs exhibited distinct growth factors and cytokine profile in the secretome. Our results unequivocally show that IFNγ and AA preconditioning of MSCs could give better therapeutic outcomes in the cell mediated therapies for COVID-19 and other inflammatory conditions.

Therapeutic Potential of Metformin-preconditioned Mesenchymal Stem Cells for Pancreatic Regeneration.

BACKGROUND: Diabetes occurs due to insulin deficiency or less insulin. To manage this condition, insulin administration as well as increased insulin sensitivity is required, but exogeneous insulin cannot replace the sensitive and gentle regulation of blood glucose levels same as ß cells of healthy individuals. By considering the ability of regeneration and differentiation of stem cells, the current study planned to evaluate the effect of metformin preconditioned buccal fat pad (BFP) derived mesenchymal stem cells (MSCs) on streptozotocin (STZ) induced diabetes mellitus in Wistar rats. MATERIALS & METHODS: The disease condition was established by using a diabetes-inducing agent STZ in Wistar rats. Then, the animals were grouped into disease control, blank, and test groups. Only the test group received the metformin-preconditioned cells. The total study period for this experiment was 33 days. During this period, the animals were monitored for blood glucose level, body weight, and food-water intake twice a week. At the end of 33 days, the biochemical estimations for serum insulin level and pancreatic insulin level were performed. Also, histopathology of the pancreas, liver and skeletal muscle was performed. RESULTS: The test groups showed a decline in the blood glucose level and an increase in the serum pancreatic insulin level as compared to the disease group. No significant change in food and water intake was observed within the three groups, while body weight was significantly reduced in the test group when compared with the blank group, but the life span was increased when compared with the disease group. CONCLUSION: In the present study, we concluded that metformin preconditioned buccal fat pad-derived mesenchymal stem cells have the ability to regenerate damaged pancreatic ß cells and have antidiabetic activity, and this therapy is a better choice for future research.

Insulin-producing cell clusters derived from human gingival mesenchymal stem cells as a model for diabetes research.

BACKGROUND: The human gingiva-derived mesenchymal stem cells (hGMSCs) possess a great potential to develop the cell-based therapy for diabetes due to its unscarred healing capacity and reparative potential. In this current study, we isolated, cultured and characterised the GMSCs and explored their potential to differentiate into Insulin Producing Cell Clusters (IPCCs). METHODS: The cells derived from gingival tissues exhibited fibroblast-like morphology. The flow cytometric analysis revealed positive expression of CD73(97.43%), CD90(95.05%), and CD105(93.17%) and negative expression of CD34(0.05%), CD45(0.09%), and HLA-DR (0.025) surface markers. We then converted this adherent fibroblast-like GMSCs into floating IPCCs using a sequential three-step protocol containing a different combination of differentiating agents. Initially, the presence of insulin in IPCCs was confirmed by dithizone staining. Glucose-stimulated insulin secretion (GSIS) assay confirmed that IPCCs secrete insulin in response to glucose. RESULTS: Generated IPCCs express pancreatic markers such as insulin, pdx1, glucagon, GLUT4 and GLUT2 as evidenced by RT-PCR analysis. Our results unequivocally showed that IPCCs can be generated from gingiva which is a potential source of postnatal MSCs. Our results offer the IPCCs generated from hGMSCs a platform for screening anti-diabetic drugs and a new autologous source of tissue for islet transplantation for the treatment of diabetes. CONCLUSIONS: Our results unequivocally demonstrate for the first time that hGMSCs can be used as an attractive non-invasive tissue source for generating IPCCs, which can be employed in diabetes research for screening antidiabetic agents and also for transplantation in type 1 diabetic patients as autologous source without the need of immunosuppression.

A novel approach to develop an animal model for oral submucous fibrosis.

Epidemiological data have proved the association of consumption of areca nut with the causation of oral submucous fibrosis (OSF). OSF is a chronic inflammatory disease with the potential for malignant transformation from 7 to 13%. The establishment of animal models makes it easier for researchers to focus on the therapeutic options to combat this disease further. We developed and compared two areca nut extract (ANE) administration methods in Swiss albino mice to establish OSF. This study compared an invasive intrabuccal injection technique with a non-invasive intraoral droplet administration. The duration of induction was around 12 weeks. Histopathology (H&E, Masson's trichrome staining) and gene expression analysis (COL-I, COL-II, and α-SMA) were performed using RT-PCR to confirm the OSF in animals. Our study showed that ANE administration through the intraoral droplet method exhibited significantly higher fibrosis than the intrabuccal injections, as evidenced by the H&E and Masson's trichrome staining. Furthermore, intraoral administration of ANE significantly upregulated the mRNA expression of COL-I, COL-II, and α-SMA, as revealed by the RT-PCR analysis. The non-invasive droplet method could simulate the absorption of areca nut seen in humans through daily dosing. This study establishes the intraoral droplet method as an efficient and non-invasive method to administer the ANE to develop OSF. These findings will aid in the efficient development of OSF animal models for interventional studies, including screening novel drugs in the reversal of the OSF.

Mesenchymal stem cell conditioned medium ameliorates diabetic serum-induced insulin resistance in 3T3-L1 cells.

BACKGROUND: Pharmacological factors used to induce insulin resistance (IR) in in vitro models may not mimic the full in vivo features of type 2 diabetes mellitus (T2DM). This study aimed to examine the ability of diabetic serum (DS) to induce IR and investigate whether adipose-derived mesenchymal stem cell conditioned medium (ADMSC-CM) reverses DS-induced IR. METHODS: DS was obtained from newly diagnosed T2DM patients. IR was induced in differentiated 3T3-L1 cells by employing dexamethasone, tumor necrosis factor alpha (TNF-α), palmitate and DS. Glucose uptake (2-[N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino]-2-deoxyglucose(2-NBDG) uptake assay), intracellular levels of reactive oxygen species (ROS), and superoxide radicals (O2-) (fluorescence microscopy and fluorometry) were analyzed in control and experimental samples. mRNA expression of key genes involved in glucose transport and inflammation were analyzed by using reverse transcription polymerase chain reaction (RT-PCR). Pro-inflammatory cytokines and phospho-insulin receptor substrate (IRS) (Ser-307) protein expression were analyzed by fluorescence activated cell sorter analysis. Statistical significance was determined by using one-way ANOVA followed by Tukey's multiple comparison tests. RESULTS: ADMSC-CM significantly increased the DS-mediated decrease in 2-NBDG uptake (11.01 ± 0.50 vs. 7.20 ± 0.30, P < 0.01) and reduced DS-driven ROS (fluorescence count, 6.35 ± 0.46 vs. 9.80 ± 0.10, P < 0.01) and O2- (fluorescence count, 3.00 ± 0.10 vs. 4.60 ± 0.09, P < 0.01) production. Further, the ADMSC-CM restored DS-induced down regulation GLUT4 (1.52-fold, P < 0.05) as well as the up-regulation of PPARγ (0.35-fold, P < 0.01), and IKKß (0.37-fold, P < 0.01) mRNA, and phospho-IRS (Ser-307) protein expression compared to the baseline (median fluorescence intensity, 88,192 ± 2720 vs. 65,450 ± 3111, P < 0.01). DS induced IR, similar to the traditionally used pharmacological factors, namely dexamethasone, TNF-α, and palmitate, which can be attributed to the significantly higher pro-inflammatory cytokines levels (TNF-α (2.28 ± 0.03 pg/mL vs. 2.38 ± 0.03 pg/mL, P < 0.01), interleukin 6 (IL)-6 (1.94 ± 0.02 pg/mL vs. 2.17 ± 0.04 pg/mL, P < 0.01), IL-17 (2.16 ± 0.02 pg/mL vs. 2.22 ± 0.002 pg/mL, P < 0.05), and interferon gamma (IFN-γ) (2.07 ± 0.02 pg/mL vs. 2.15 ± 0.04 pg/mL, P < 0.05)) in DS. CONCLUSIONS: DS can be explored as a novel inducer of IR in in vitro studies with further standardization, substituting the conventionally used pharmacological factors. Our findings also affirm the validity of ADMSC-CM as a prospective insulin sensitizer for T2DM therapy.

Pre-conditioning of Mesenchymal Stem Cells with Piper longum L. augments osteogenic differentiation.

ETHNOPHARMACOLOGICAL RELEVANCE: The Indian Traditional Medicine, Ayurveda prescribes Piper longum L. popularly known as Long Pepper (Pippali) for the treatment of inflammatory and degenerative diseases. Therapeutic benefits of Piper longum L. are mainly attributed to the anti-inflammatory and arthritic potential. AIM OF THE STUDY: This study was aimed to explore the activity of Piper longum L. fruit extract on proliferation and osteogenic differentiation of human Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) to find out it's possible role as anti-osteoporotic agent. MATERIALS AND METHODS: Proliferation of WJMSCs treated with Piper longum L. fruit extract was assessed by MTT assay and Cell Cycle Analysis. Effect of Piper longum L. preconditioning on osteogenic differentiation was performed. Ca2+ accumulation and matrix mineralization (Von Kossa and Alizarin Red Staining), alkaline phosphatase (ALP) activity and gene expression of key mRNA (RT PCR) was analyzed. RESULTS: Significant increase in the proliferation of WJMSCs was observed upon treatment of Piper longum L. at 5 µg/mL (P < 0.001) which can be attributed to the significant decrease in apoptotic cells (P < 0.05) as evidenced by cell cycle analysis. Preconditioning of Piper longum L. (10-100 µg/mL) enhanced Ca2+ accumulation and matrix mineralization as observed by Von Kossa and Alizarin Red staining where ALP activity was elevated 3.6 folds as compared to untreated WJMSCs (P < 0.001). RT-PCR analysis exhibited up regulation of Runx2, Osterix, ALP and OPN mRNAs. CONCLUSIONS: We demonstrate for the first time that Piper longum L. fruit extract enhanced osteogenic differentiation of WJMSCs. This finding can be clinically translated into development of an anti-osteoporotic agent.

Human postnatal Mesenchymal Stem Cell Derived Islets as a Model for Diabetes Research.

The scarcity of human cadaver islets for transplantation in patients with Diabetes Mellitus (DM) has necessitated the search for alternative islet sources. With advancing islet biology research, Islet-Like Clusters (ILCs) derived from stem cells have demonstrated potential for treating DM and in novel drug discovery programs for drug and cytotoxicity testing. In vitro differentiation of ILCs from stem cells also provides an opportunity to mimic the in vivo islet developmental pathways. In vitro derived ILCs are often considered immature as they do not respond to glucose challenges efficiently. However, the in vitro and in vivo performance of ILCs can be improved by pharmacological preconditioning. In this review, we discuss how ILCs generated from human postnatal tissues can be utilized as an in-vitro model to study cytotoxicity, drug screening and enhancement of transplantation efficacy. The use of human cadaver islets is not permitted for research purposes in India. Under these restrictions, the application of ILCs in drug screening and their role in complementing, reducing, and replacing animal testing will evolve as a reliable platform for in vitro screening as well as for stem cell-based treatment in DM.

Hypothesizing the therapeutic potential of mesenchymal stem cells in oral submucous fibrosis.

Oral submucous fibrosis is the direct consequence of a sustained pro-inflammatory environment characterized by excessive collagen deposition causing tissue fibrosis, and progressive degeneration of vital structures including muscle. The pathogenesis of oral submucous fibrosis is largely mediated by the pro-inflammatory, pro-fibrotic cytokines, excessive oxidative stress, abnormal angiogenesis, and epithelial to mesenchymal transition. Mesenchymal stem cells largely known for their regenerative potential have shown to have an immunomodulatory, anti-fibrotic, anti-oxidative, and angiogenic potential. Thus, mesenchymal stem cells, when introduced in an oral submucous fibrosis micro-environment, could potentially counter the progressive fibrosis. The present hypotheses discuss the various pathogenic aspects of oral submucous fibrosis and the properties of mesenchymal stem cells which could aid in halting the disease progression.

Conditioned medium of adipose derived Mesenchymal Stem Cells reverse insulin resistance through downregulation of stress induced serine kinases.

Insulin resistance (IR) is a constituent part of Type 2 Diabetes Mellitus (T2DM). Conditioned medium from Adipose derived Mesenchymal Stem Cells (ADMSCs-CM) has been shown to reverse IR. However, its effect on cellular stress is not well established. The objective of this study was to explore the effect of ADMSCs-CM on reactive oxygen species, mitochondrial membrane potential (ΔΨm), endoplasmic reticulum (ER) stress and expression of oxidative and inflammatory stress induced serine kinases (SISK) which are pathophysiologically linked to IR. In insulin resistant, 3T3-L1 adipocytes and C2C12 myoblast cell culture models, glucose uptake was assayed by 2-NBDG uptake. Immunomodulatory cytokines, intracellular reactive oxygen species generation, ΔΨm and protein expression of JNK1, IKKß and phospho-IRS1 (307) were analyzed using FACS. mRNA expression of ER stress markers (CHOP1 and IRE1) and SISK (JNK1, IKKß, ERK1 and S6K1) were analyzed using RT-PCR. ADMSCs-CM effectively improve glucose uptake as evidenced by 2-NBDG uptake assay. FACS analysis showed that ADMSCs-CM possessed significantly higher levels of IL-6 and IL-10. ADMSCs-CM decreased intracellular generation of reactive oxygen species where it restored ΔΨm in C2C12 cells. ADMSCs-CM mediated reduction in ER stress was confirmed by down-regulation in CHOP1 and IRE1 mRNA expression. ADMSCs-CM treatment showed significant down-regulation of SISK mRNA expression including IKKß, JNK, ERK and S6K1. Our results unequivocally demonstrate for the first time the mechanism of action of ADMSCs-CM in amelioration IR by reducing oxidative and inflammatory cellular stress. This study identifies SISK as potential therapeutic targets for T2DM therapy.

Herbal pre-conditioning induces proliferation and delays senescence in Wharton's Jelly Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are multipotent stem cells which are being explored for various clinical applications. Isolation and in-vitro expansion of MSCs remain important in achieving desired cell number for the therapy. However, in-vitro proliferation of MSCs is often associated with senescence and early onset of apoptosis which limits its therapeutic ability and long term clinical use. Tinospora cordifolia and Withania somnifera are used widely in Ayurveda: the traditional Indian system of medicine and are reported to have rejuvenating and anti-aging potential. In the present study, we investigated the effect of Tinospora cordifolia and Withania somnifera on proliferation and senescence of wharton's jelly MSCs (WJMSCs) in-vitro. METHODS: WJMSCs were treated in culture medium with Tinospora cordifolia leaf and Withania somnifera root extracts to examine their effect on proliferation and senescence properties of WJMSCs. Proliferation of WJMSCs was assayed by cell count, MTT, BrdU incorporation assay, cell cycle analysis and Ki67 mRNA expression. Senescence was demonstrated using ß-galactosidase senescence assay and associated mRNA markers. RESULTS: Culture medium supplemented with Tinospora cordifolia leaf and Withania somnifera root extracts exhibited significant increase in proliferation of WJMSCs as evidenced by cell count and MTT assay. Cell cycle analysis using propidium iodide showed increase in G2/M phase and decrease in apoptotic cells. BrdU incorporation and upregulation of proliferation marker ki67 by RT PCR showed increased DNA synthesis/proliferation in Tinospora cordifolia and Withania somnifera extract treated MSCs. Delayed senescence was confirmed by ß-galactosidase senescence assay and down regulation of senescence marker p21. CONCLUSION: Our results demonstrate for the first time that Tinospora cordifolia and Withania somnifera extracts support proliferation and inhibit senescence in WJMSCs making them suitable candidates as supplements for in-vitro expansion without affecting the cell viability indicating its non-toxic nature.