Postbiotics of Naturally Fermented Synbiotic Mixture of Rice Water Aids in Promoting Colonocyte Health.

The eubiotic state of the gut microbiota is primarily brought about by various probiotic species that colonize the gut. It is becoming very clear that the probiotic-metabolite mixtures in the gut luminal milieu is central in establishing cross-kingdom signalling networks to maintain gut-multi-organ axes health. Culturally, different fermented foods and beverages have been regional staples since ancient times, and are known to be enriched with probiotics. However, regional variations including the environment, the staple food source (prebiotics), and fermentation methods, among other factors, influence the fermenting probiotic species. Fermented rice water (FRW), an economical, easy to make, simple beverage is a rich source of synbiotics. Therefore, consumption of fermented rice water allows for the intake of a variety of region-specific live probiotics. The secondary metabolites (postbiotics) present in such symbiotic mixtures may also contribute toward maintaining normal intestinal cellular functions. In this study, we highlight that regional staples such as rice consumed in their fermented form may hold promise in alleviating gut-related diseases. Our results show that simple overnight fermentation of cooked edible rice enables the growth of probiotic bacterial species belonging to the Lactic Acid Bacteria group (Leuconostoc lactis, Weisella confusa, Weisella cibacria, Lactococcus lactis, lactococcus taiwanensis, Lactobacillus fermentum, Lactobacillus nagelii, and Lactobacillus delbrueckii ssp. indicus). Metabolomic analysis of the overnight fermented and over two-nights fermented rice water identified more than 200 postbiotic metabolites. Our results show that postbiotics contributing to energy metabolism, gut-multiorgan axes, and microbial paraprobiotics are enriched in the overnight (~10 h) fermented rice water as compared to the over two-nights fermented rice water. Functional analysis via gene expression studies for nutrient absorption (mct-1 and mct-2) and barrier integrity (occludin and zo-1) reveals significant upregulation of these genes upon FRW treatment of HT29 colon cells. This study is a first-of-its-kind to demonstrate the proof-of-principle that postbiotics of naturally fermented rice water positively modulates colonocyte health.

Comparison of mammosphere formation from stem-like cells of normal breast, malignant primary breast tumors, and MCF-7 cell line.

BACKGROUND: Mammosphere formation assay has become a versatile tool to quantify the activity of putative breast cancer stem cells in non-adherent in vitro cultures. However, optimizing the suspension culture system is crucial to establish mammosphere cultures from primary breast tumors. METHODS: This study aimed at determining the self-renewal and sphere-forming potential of breast cancer stem-like cells derived from human primary invasive ductal carcinoma and normal breast tissue samples, and MCF-7 breast cancer cell line using an optimal suspension culture system. Mammosphere-forming efficiency of the mammospheres generated from the tissue samples and cell line were compared. We evaluated the expression of CD44+/CD24-/low and CD49f+/EpCAM-/low phenotypes in the stem-like cells by flow cytometry. CK-18, CK-19, α-SMA, and EpCAM marker expression was assessed using immunohistochemical staining. RESULTS: Breast epithelial cells isolated from the three samples formed two-dimensional spheroids in suspension cultures. Interestingly, mammospheres formed from patient-derived primary breast tumors were enriched in breast cancer stem-like cells with the phenotype CD44+/CD24-/low and exhibited a relatively more number of large spheres when compared to the normal breast stem cells. MCF-7-derived SCs were more aggressive and resulted in the formation of a significantly higher number of spheroids. The expression of CK-18/CK-19 and α-SMA/EpCAM proteins was confirmed in breast cancer tissues. CONCLUSIONS: Thus, the use of primary tumor specimens and breast cancer cell lines as suitable models for elucidating the breast cancer stem cell activity was validated using mammosphere culture system.

Effect of Cell Derived Matrices on growth and differentiation of human Wharton's jelly derived Mesenchymal Stem Cells.

Cell derived matrices (CDMs) are scaffolds constructed by decellularization of cellular matrices from different tissues and organs. Since cell derived matrices mimic the ECM of native tissues, CDM plays an essential role in the preparation of bioscaffolds. CDM scaffolds from Mesenchymal Stem Cells (MSCs) have been reported to support cell adhesion and proliferation of its own cells. Therefore, in this study we aimed to test if growth of human Wharton's jelly derived MSCs (hWJ-MSCs) may be enhanced when cultured on their own cell derived matrices. To do this, MSCs were induced to generate ECM using ascorbic acid. Thus, obtained matrices were decellularized and characterized quantitatively for changes in their biochemical components (total protein, collagen, glycosaminoglycans) and qualitatively for fibronectin, laminin and collagen (I & IV) by immunostaining. Our results show the retention of essential ECM components in the decellularized WJ-CDM. The influence of WJ-MSC-derived CDM on proliferation and differentiation of WJ-MSCs were evaluated by comparing their growth on collagen and fibronectin only coated plates. A non-coated tissue culture polystyrene plate (TCPS/WC) served as control. Our cell proliferation results show that no significant changes were observed in the proliferation of MSCs when cultured on WJ-MSC derived CDM as compared to the bio-coated and non-coated cultures. However, gene expression analysis of the differentiation process showed that osteogenic and adipogenic differentiation potential of the WJ-MSCs was significantly increased upon culturing them on WJ-MSC-CDM. In conclusion, the present study reveals that the WJ-MSCs cultured on WJ-MSC-CDM may augment osteogenic and adipogenic differentiation.

Differential immunomodulation of human mesenchymal stromal cells from various sources in an inflammation mimetic milieu.

Mesenchymal stromal cells (MSCs) are very advantageous in the field of regenerative medicine because of their immunomodulatory properties. However, reports show that these properties vary from source to source. Hence, understanding the source-dependent specificity of MSCs and their immunomodulatory abilities will enable optimal use of MSCs in cell-based therapies. Here, we studied human MSCs from three different sources, adipose tissue (AT), bone marrow (BM) and Wharton's jelly (WJ), with respect to phenotypic responses of human peripheral blood mononuclear immune cells (hPBMCs/MNCs) and the concurrent changes in cytokine expression in MSCs, under mitogen-stimulated co-culture conditions. We used cytometric analysis to study the immunoregulatory properties of MSCs on MNCs and cytokine profiling of MSCs using a customized PCR array and solid-phase sandwich enzyme-linked immunosorbent assay. Our results reveal differential modulation of immune cells as well as MSCs upon activation by the mitogen phytohemagglutinin, independently and in co-culture. Notably, we observed source-specific MSC-cytokine signatures under stimulated conditions. Our results show that AT-MSCs up-regulate VEGF, BM-MSCs up-regulate PTGS-2 and WJ-MSCs increase expression of IDO considerably compared with controls. This remarkable modulation in source-specific cytokine expression was also validated at a functional level by quantitative protein expression studies. In our hands, even though MSCs from AT, BM and WJ sources exhibit characteristic immunomodulatory properties, our results highlight that MSCs sourced from different tissues may exhibit unique cytokine signatures and thus may be suitable for specific regenerative applications.

A Clinical Study to Evaluate Autofluorescence Imaging of Diabetic Foot Ulcers Using a Novel Artificial Intelligence Enabled Noninvasive Device.

Diabetic foot ulcers, with worldwide prevalence ranging from 12%-25%, are an important cause of nontraumatic lower limb amputation. Evidence-based assessment of early infection can help the clinician provide the right first line treatment thus helping improve the wound closure rate. Illuminate®, a novel point of care device working on multispectral autofluorescence imaging, helps in the rapid identification and classification of bacteria. This study was aimed to evaluate the diagnostic accuracy of the device in detecting bacterial gram type against standard culture methods. A total of 178 patients from a tertiary care center for diabetes was recruited and 203 tissue samples were obtained from the wound base by the plastic surgeon. The device was handled by the trained investigator to take wound images. The tissue samples were taken from the color-coded infected region as indicated by the device's Artificial Intelligence algorithm and sent for microbial assessment. The results were compared against the Gram type inferred by the device and the device was found to have an accuracy of 89.54%, a positive predictive value of 86.27% for detecting Gram-positive bacteria, 80.77% for Gram-negative bacteria, and 91.67% for no infection. The negative predictive value corresponded to 87.25% for Gram-positive, 92% for Gram-negative, and 96.12% for no infection. The Results exhibited the accuracy of this novel autofluorescence device in identifying and classifying the gram type of bacteria and its potential in significantly aiding clinicians towards early infection assessment and treatment.

Effect of Human Platelet Lysate in Differentiation of Wharton's Jelly Derived Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are highly preferred in clinical therapy for repair and regeneration of diseased tissues for their multipotent properties. Conventionally, MSCs have been cultured in media supplemented with animal derived serum, however, it is ideal to expand MSCs in media containing supplements of human origin for clinical therapy. Currently, a number of human derived products are being studied as an alternative to animal sources. Amongst these, platelet lysate (PL) has gained interest in the culture of MSCs without affecting their phenotypic property. OBJECTIVE: In this study, we used various concentration of PL (2.5, 5, 7.5 & 10%) in the growth medium of MSCs to identify the least concentration of PL that could be an effective alternative to animal products. METHODS: MSCs were isolated from Wharton's Jelly by using explant method and expanded in various concentration of PL supplemented medium against the standard FBS containing medium. WJ-MSCs were characterised as per the minimal criteria proposed by International Society for Cell therapy (ISCT), Proliferation study by BrdU assay, gene expression study by qRT-PCR, sterility test for bacteria, Mycoplasma by PCR and endotoxin detection by LAL assay. RESULTS: Whartons jelly derived MSCs (WJ-MSCs) cultured using standard medium supplemented with various concentration of PL exhibited enhanced proliferation and differentiation potential, unaltered immunophenotypic property and genetic stability when compared with the commercial medium containing 10% FBS. CONCLUSION: The least concentration of PL for an ideal expansion of MSCs was found to be 2.5% and was comparable to FBS.

Role of Human Wharton's Jelly Derived Mesenchymal Stem Cells (WJ-MSCs) for Rescue of d-Galactosamine Induced Acute Liver Injury in Mice.

BACKGROUND/AIM: Mesenchymal stem cells (MSCs) are multipotent precursor cells having self-renewal ability making them a candidate for use in regenerative medicine. Acute liver injury results in sudden loss of hepatic function leading to organ failure. Liver transplantation is often required to salvage patients with acute liver failure. Due to shortage of organs, identification of alternate method is the need of the hour. In view of this, an attempt has been made to check the regenerative ability of WJ-MSCs (wharton's jelly derived MSC) in mice models for acute liver injury. METHODS: Swiss albino mice weighing 25 ± 5 g were used in this study. The control mice (Group I), was given saline. Group II mice received d-Galactosamine (d-GalN-800 mg/kg; i.p). Group III mice similar with Group II, received WJ-MSCs (5 × 105 cells/0.5 ml DMEM) through tail vein, 24 h after d-GalN administration and Group IV mice received MSC alone. RESULTS: Parameters, indicative of hepatotoxicity and oxidative stress were analyzed. A two-fold elevation in the marker enzymes of liver toxicity such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (SAP), and total serum bilirubin (TBIL) confirms hepatocellular injury, while a greater than four-fold increase in malondialdehyde (MDA) formation, along with around 40% fall in superoxide-dis-mutase (SOD) activity was indicative of oxidative stress and loss of hepatocellular membrane integrity induced by d-GalN. The above biochemical and pathological changes were significantly restored in mice that received WJ-MSCs indicating hepatoprotective and probable regenerative property. CONCLUSION: The present study showed that WJ-MSC treatment is able to rescue/ameliorate the hepatotoxicity induced by d-GalN in mice.