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Background: Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells. Materials and Methods: Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 370C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 370C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2. Results: In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 103/μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2. Conclusion: Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Background: Stem cells, including neural stem cells (NSCs), are endowed with self-renewal capability and hence hold great opportunity for the institution of replacement/protective therapy. We propose a method for in vitro generation of stromal cells from human adipose tissue and their differentiation into neural cells.Materials and Methods: Ten grams of donor adipose tissue was surgically resected from the abdominal wall of the human donor after the participants' informed consents. The resected adipose tissue was minced and incubated for 1 hour in the presence of an enzyme (collagenase-type I) at 370C followed by its centrifugation. After centrifugation, the supernatant and pellets were separated and cultured in a medium for proliferation at 370C with 5% CO2 for 9-10 days in separate tissue culture dishes for generation of mesenchymal stromal cells (MSC). At the end of the culture, MSC were harvested and analyzed. The harvested MSC were subjected for further culture for their differentiation into neural cells for 5-7 days using differentiation medium mainly comprising of neurobasal medium. At the end of the procedure, culture cells were isolated and studied for expression of transcriptional factor proteins: orthodenticle homolog-2 (OTX-2), beta-III-tubulin (β3-Tubulin), glial-fibrillary acid protein (GFAP) and synaptophysin-β2.Results: In total, 50 neural cells-lines were generated. In vitro generated MSC differentiated neural cells' mean quantum was 5.4 ± 6.9 ml with the mean cell count being, 5.27 ± 2.65 × 103/μl. All of them showed the presence of OTX-2, β3-Tubulin, GFAP, synaptophysin-β2.Conclusion: Neural cells can be differentiated in vitro from MSC safely and effectively. In vitro generated neural cells represent a potential therapy for recovery from spinal cord injuries and neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Transplantation tolerance is still a Utopian dream for many transplanters. Mesenchymal stem cells (MSC) have shown immuno-modulatory and tolerogenic effects in experimental models. We present a 29-year-old male with end stage renal disease (ESRD) who was transplanted with HLA 4/6 matched kidney from 51-year-old father in June 2010 preceded by co-infusion of donor-adipose tissue derived mesenchymal stem cells (AD-MSC) and bone marrow derived hematopoietic stem cells (BM-HSC) under non-myeloablative conditioning for deleting rejecting T and B-cells. He has maintained fairly stable graft function with serum creatinine (SCr) between 1.5 and 1.8 mg/dL at 3 years post-transplant with absence of donor specific antibodies (DSA), normal protocol graft biopsy, and peripheral T-regulatory cell levels (pTregs) (CD127low/−CD25highCD4+) of 4.57% on zero immunosuppression since 6 months. [ABSTRACT FROM AUTHOR]

Background: Diabetics are incapable of producing insulin/have autoimmune mechanisms making it ineffective to control glucose secretion. We present a prospective study of glucose-sensitive insulin-secreting mesenchymal stem cells (IS-MSC) generated from human adipose tissue (h-AD) sans xenogenic material. Materials and Methods: Ten grams h-AD from donor anterior abdominal wall was collected in proliferation medium composed of α-Minimum Essential Media (α-MEM), albumin, fibroblast-growth factor and antibiotics, minced, incubated in collagenase-I at 37°C with shaker and centrifuged. Supernatant and pellets were separately cultured in proliferation medium on cell+ plates at 37°C with 5% CO2 for 10 days. Cells were harvested by trypsinization, checked for viability, sterility, counts, flow-cytometry (CD45-/90+/73+), and differentiated into insulin-expressing cells using medium composed of DMEM, gene expressing up-regulators and antibiotics for 3 days. They were studied for transcriptional factors Pax-6, Isl-1, pdx-1 (immunofluorescence). C-peptide and insulin were measured by chemiluminescence. In vitro glucose sensitivity assay was carried out by measuring levels of insulin and C-peptide secretion in absence of glucose followed by 2 hours incubation after glucose addition. Results: Mean IS-AD-MSC quantum was 3.21 ml, cell count, 1.5 x10³ cells/µl), CD45-/90+/73+ cells were 44.37% /25.52%. All of them showed presence of pax-6, pdx-1, and Isl-1. Mean C-Peptide and insulin levels were 0.36 ng/ml and 234 µU/ml, respectively, pre-glucose and 0.87 ng/ml and 618.3 µU/ml post-glucose additions. The mean rise in secretion levels was 2.42 and 2.65 fold, respectively. Conclusion: Insulin-secreting h-AD-MSC can be generated safely and effectively showing in vitro glucose responsive alteration in insulin and C-peptide secretion levels. [ABSTRACT FROM AUTHOR]

Transplantation using immunosuppression/induction therapies has controlled acute rejections; however, there is no answer for chronic graft attrition. Donor-specific antibodies (DSA) are believed to cause antibody mediated rejections eventually causing chronic graft loss. Regulatory T cells (T-regs) are believed to protect the graft from immune injury. We report a 53-year-old woman transplanted with her son's kidney using donor-specific transfusion and stem cells (SC) under non-myeloablative conditioning of cyclophosphamide, anti-T and anti-B-cell antibodies and Bortezomib. The patient was on low-dose steroid monotherapy under immune monitoring of DSA and serum creatine. Graft biopsy at 1 and 3.5 years post-transplant was unremarkable in spite of the presence of DSA. Peripheral T-regs (pTregs) at 3.5 years post-transplant were 3.54%. This case shows that DSA are not necessarily detrimental to the renal allograft. We further hypothesise that pTregs were induced from SC and sustained to protect this graft from cytotoxic T cells and DSA. [ABSTRACT FROM AUTHOR]

The authors report a 17-year-female and a 19-year-male with uncontrolled insulin-dependent diabetes mellitus (IDDM) for ⩾10 years, treated with insulin-secreting human adipose tissue derived mesenchymal stem cells (IS-h-ADMSC). Both had hypothyroidism and were diagnosed as polyglandular autoimmune syndrome type-3 (PGAS-3). PGAS are rare polyendocrinopathies with ⩾2 endocrine disorders mediated by autoimmune mechanisms leading to hypo-function and organ failure. Therapeutic options are hormone replacement, immunosuppression and avoiding infection. The authors administered autologous H-AD-IS-MSC+bone marrow-derived haematopoietic stem cells (HSC) into portal circulation with conditioning of cyclophosphamide, bortezomib, rituximab and rabbit-antithymoglobulin. Over follow-up of 38 and 16 months, respectively, both are doing well with sustained fall of glycosylated haemoglobin (Hb1Ac) from 8.1 to 6.4% and 14.2 to 8.6%, respectively and C-peptide raised from 0.01 to 0.23 ng/ml and 0.1 to 0.34 ng/ml, respectively with sustained 40% decreased insulin requirement. Thus long-term control of IDDM in PGAS-3 with co-transplantation of H-AD-IS-MSC+HSC can be achieved safely and effectively. [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) is a common cause of end-stage renal disease (ESRD). Various factors contribute to wide fluctuations in blood glucose levels and exogenous insulin requirement in such patients even after renal transplantation (RT). Simultaneous pancreas-kidney transplantation is one of the therapies for these patients. Stem cell (SC) therapy for T1DM and for minimisation of immunosuppression after RT has shown encouraging results. We report a 30-year-old-man with T1DM since 15 years and ESRD since 2 years, who underwent living donor RT and co-infusion of in vitro generated insulin-making cells differentiated from donor adipose tissue derived mesenchymal stem cells and bone marrow -derived haematopoietic SC into subcutaneous tissue, portal and thymic circulation under non-myeloablative conditioning. Over follow-up of 13 months he has stable graft function with serum creatinine, 1.2 mg/dl, zero rejection and glycosylated haemoglobin level of 6.1% on calcineurin-inhibitor based therapy. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) is not likely to recover by current therapeutic modalities. Stem cell (SC) therapy (SCT) has promising results in regenerative medicine. We present our experience of co-infusion of autologous adipose tissue derived mesenchymal SC differentiated neuronal cells (N-Ad-MSC) and hematopoietic SCs (HSCs) in a set of patients with posttraumatic paraplegia., Materials and Methods: Ten patients with posttraumatic paraplegia of mean age 3.42 years were volunteered for SCT. Their mean age was 28 years, and they had variable associated complications. They were subjected to adipose tissue resection for in vitro generation of N-Ad-MSC and bone marrow aspiration for generation of HSC. Generated SCs were infused into the cerebrospinal fluid (CSF) below injury site in all patients., Results: Total mean quantum of SC infused was 4.04 ml with a mean nucleated cell count of 4.5 × 10(4)/μL and mean CD34+ of 0.35%, CD45-/90+ and CD45-/73+ of 41.4%, and 10.04%, respectively. All of them expressed transcription factors beta-3 tubulin and glial fibrillary acid protein. No untoward effect of SCT was noted. Variable and sustained improvement in Hauser's index and American Spinal Injury Association score was noted in all patients over a mean follow-up of 2.95 years. Mean injury duration was 3.42 years against the period of approximately 1-year required for natural recovery, suggesting a positive role of SCs., Conclusion: Co-infusion of N-Ad-MSC and HSC in CSF is safe and viable therapeutic approach for SCIs.

Introduction: We evaluated donor hypo-responsiveness in renal allograft recipients to donor adipose tissue-derived mesenchymal stem cell (h-AD-MSC) +hematopoietic stem cell transplantation (HSCT) vs. HSCT alone., Methods: Patients were divided into 2 demographically equal groups (n = 100) A and B subjected to equal non-myeloablative conditioning of target-specific irradiation, anti-T + B cell antibodies and cyclophosphamide with HSCT. Group A was administered h-AD-MSC additionally. Transplantation was performed following favorable cross-matching. Cyclosporine, 3 mg/kg BW/day + prednisone, 20 mg/day were immunosuppressants for first 3 months, cyclosporine was replaced by azathioprine subsequently and prednisone lowered to 5-10 mg/day. Peripheral blood chimerism (PBC) was studied using fluorescent in situ hybridization technique at 3/18 months post transplant. Biopsy was performed for graft dysfunction and reported as per Banff criteria,'05., Results: Mean nucleated HSC counts (n × 10(8)/kgBW) was 7.32 with mean CD34+ yield 0.09% in group A; and 6.98 and 0.40% in group B, respectively; CD45-/90+ was 13.49% in former. Over 18 months post transplant, former had mean serum creatinine (SCr), 1.59 mg%, 12% acute rejection (AR) episodes, 3% patient, 1% patient +graft loss; latter had mean SCr 1.49 mg%, 18% AR episodes, 1% patient, 6% graft and 8% patient +graft losses. PBC was higher (4%) in former than later (1.8%)., Conclusion: Combined h-AD-MSC +HSCT under non-myeloablative conditioning was safe, more effective than HSCT alone to achieve donor hypo-responsiveness with adequate stable graft function and reduced rejection episodes.