Sachin S. Kadam

HUMAN PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND ISLET-LIKE CELL CLUSTERS GENERATED FROM THESE CELLS AS A NOVEL SOURCE FOR STEM CELL THERAPY IN DIABETES

Placental tissue holds great promise as a source of cells for regenerative medicine due to its plasticity, and easy availability. Human placenta-derived mesenchymal stem cells (hPDMSCs) have the potential to differentiate into insulin-producing cells. Upon transplantation, they can reverse experimental diabetes in mice. However, it is not known whether culture-expanded undifferentiated hPDMSCs are capable of restoring normoglycemia upon transplantation in streptozotocin (STZ)-induced diabetic mice. Hence we prepared long-term cultures of hPDMSCs from the chorionic villi of full-term human placenta. Flow cytometry analyses and immunocytochemistry study revealed bonafide mesenchymal nature of the isolated hPDMSCs. These cultures could differentiate into adipogenic, oesteogenic, chondrogenic, and neuronal lineages on exposure to lineage-specific cocktails. Furthermore, we showed that hPDMSCs can form islet-like cell clusters (ILCs) on stepwise exposure to serum-free defined media containing specific growth factors and differentiating agents. qRT-PCR showed the expression of insulin, glucagon, and somatostatin in undifferentiated hPDMSCs and in ILCs. Differentiated ILCs were found to express human insulin, glucagon, and somatostatin by immunocytochemistry. Additionally, ILCs also showed abundance of pancreatic transcription factors ngn3 and isl1. Both undifferentiated hPDMSCs and ILCs exihibited insulin secretion in response to glucose. Transplantation of hPDMSCs or ILCs derived from hPDMSCs in STZ-induced diabetic mice led to restoration of normoglycemia. Our results demonstrate, for the first time, reversal of hyperglycemia by undifferentiated hPDMSCs and ILCs derived from hPDMSCs. These results suggest human placenta-derived MSCs as an alternative source for cell replacement therapy in diabetes.

Human breast milk is a rich source of multipotent mesenchymal stem cells

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be “reprogrammed” to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.

The biocompatibility and separation performance of antioxidative polysulfone/vitamin E TPGS composite hollow fiber membranes.

The extended interaction of blood with certain materials like hemodialysis membranes results in the activation of cellular element as well as inflammatory response. This results in hypersensitive reactions and increased reactive oxygen species, which occurs during or immediately after dialysis. Although polysulfone (Psf) hollow fiber has been commercially used for acute and chronic hemodialysis, its biocompatibility remains a major concern. To overcome this, we have successfully made composite Psf hollow fiber membrane consisting of hydrophilic/hydrophobic micro-domains of Psf and Vitamin E TPGS (TPGS). These were prepared by dry-wet spinning using 5, 10, 15, 20 wt% TPGS as an additive in dope solution. TPGS was successfully entrapped in Psf hollow fiber, as confirmed by ATR-FTIR and TGA. The selective skin was formed at inner side of hollow fibers, as confirmed by SEM study. In vitro biocompatibility and performance of the Psf/TPGS composite membranes were examined, with cytotoxicity, ROS generation, hemolysis, platelet adhesion, contact and complement activation, protein adsorption, ultrafiltration coefficient, solute rejection and urea clearance. We show that antioxidative composite Psf exhibits enhanced biocompatibility, and the membranes show high flux and high urea clearance, about two orders of magnitude better than commercial hemodialysis membranes on a unit area basis.

Islet neogenesis from the constitutively nestin expressing human umbilical cord matrix derived mesenchymal stem cells

The scarcity of islets for transplantation calls for an alternative sources of islets. The human umbilical cord has been shown to be a reservoir of multipotent stem cells with capacity to differentiate into ectodermal, mesodermal and endodermal lineages. The present investigation deals with isolation and characterization of mesenchymal stem sells (MSC) derived from human umbilical cord and their differentiation into functional islets. Since these MSCs were found to constitutively express nestin we hypothesized that these would be ideal candidates for islet neogenesis without any further manipulation. Human umbilical cord matrix stem cells (hUCMSCs) were found to express CD29, CD44, CD73, CD90, CD105, smooth muscle actin, nestin, vimentin, proliferation marker Ki67 and embryonic markers Oct4, SSEA4. These were found to be negative for CD33, CD34, CD45 and HLA DR. Human UCMSCs exhibited high proliferating capacity for extended period indicating potential for scaling up. When subjected to a cocktail of specific differentiating factors, these cells differentiated into fat, cartilage, bone, neurons and islet like clusters (ILCs). These ILCs stained positive for diphenylthiocarbazone (DTZ) and expressed human C-peptide, insulin and glucagon. Real time qPCR analysis of newly generated islets further demonstrated abundance of Pdx-1, Ngn3, insulin, glucagon and somatostatin transcripts. On transplantation in experimental diabetic mice these ILCs restored normoglycemia, body weight and exhibited normal glucose tolerance test indicating their functional status. Thus, the present study demonstrates potential of constitutively expressing nestin positive progenitor from umbilical cord as a novel source for islet neogenesis and their usage in cell replacement therapy for diabetes.

Reversal of experimental diabetes in mice by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells using immuno-isolatory macrocapsules

BACKGROUND AIMSnThe ethical and biologic limitations with current sources of stem cells have resulted in a quest to look for alternative sources of multipotent stem cells of human origin. Amniotic membrane is of interest as a source of cells for regenerative medicine because of its ease of availability, plasticity and inexhaustible source that does not violate the sanctity of independent life. Although researchers have shown the stem cell-like potential of human amniotic epithelial cells, the mesenchymal part of amnion has remained less explored.nnnMETHODSnWe established a long-term culture of mesenchymal-like stem cells derived from full-term human amniotic membrane and their differentiation into functional pancreatic lineage.nnnRESULTSnThe amnion-derived mesenchymal-like stem cells expressed various mesenchymal markers and demonstrated multilineage differentiation capacity. We also observed that these cells could form islet-like clusters (ILC) on exposure to serum-free defined media containing specific growth factor and differentiating agents. Differentiated ILC showed expression of human insulin, glucagon and somatostatin by immunocytochemistry, while quantitative reverse transcription/real-time-polymerase chain reaction (qRT-PCR) data demonstrated the expression of insulin, glucagon, somatostatin, Ngn3 and Isl1. Moreover, encapsulation of the ILC in polyurethane-polyvinyl pyrrolidone macrocapsules and their subsequent transplantation in experimental diabetic mice resulted in restoration of normoglycemia, indicating their ability to respond to high glucose without immunorejection.nnnCONCLUSIONSnOur results demonstrate that amnion-derived mesenchymal stromal cells can undergo islet neogenesis, indicating amnion as an alternative source of islets for cell replacement therapy in diabetes.

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nanofibers over hydroxyapatite: a comparative study.

The aim of this study was to compare physico-chemical and biological properties of hydroxyapatite (HA) and hardystonite (HS) based composite scaffolds. Hardystonite (Ca2ZnSi2O7) powders were synthesized by a sol-gel method while polycaprolactone-hardystonite (PCL-HS) and polycaprolactone-hydroxyapatite (PCL-HA) were fabricated in nanofibrous form by electrospinning. The physico-chemical and biological properties such as tensile strength, cell proliferation, cell infiltration and alkaline phosphatase activity were determined on both kinds of scaffolds. We found that PCL-HS scaffolds had better mechanical strength compared to PCL-HA scaffolds. Addition of HA and HS particles to PCL did not show any inhibitory effect on blood biocompatibility of scaffolds when assessed by hemolysis assay. The in vitro cellular behavior was evaluated by growing murine adipose-tissue-derived stem cells (mE-ASCs) over the scaffolds. Enhanced cell proliferation and improved cellular infiltrations on PCL-HS scaffolds were observed when compared to HA containing scaffolds. PCL-HS scaffolds exhibited a significant increase in alkaline phosphatase (ALP) activity and better mineralization of the matrix in comparison to PCL-HA scaffolds. These results clearly demonstrate the stimulatory role of Zn and Si present in HS based composite scaffolds, suggesting their potential application for bone tissue engineering.

Generation of functional islets from human umbilical cord and placenta derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, mesenchymal stem cells (MSCs) derived from other adult tissue sources have been considered as an alternative. The human umbilical cord and placenta are easily available noncontroversial sources of human tissue, which are often discarded as biological waste, and their collection is noninvasive. These sources of MSCs are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs derived from umbilical cord and placenta are multipotent and have the ability to differentiate into various cell types crossing the lineage boundary towards endodermal lineage. The aim of this chapter is to provide a detailed reproducible cookbook protocol for the isolation, propagation, characterization, and differentiation of MSCs derived from human umbilical cord and placenta with special reference to harnessing their potential towards pancreatic/islet lineage for utilization as a cell therapy product. We show here that mesenchymal stromal cells can be extensively expanded from umbilical cord and placenta of human origin retaining their multilineage differentiation potential in vitro. Our report indicates that postnatal tissues obtained as delivery waste represent a rich source of mesenchymal stromal cells, which can be differentiated into functional islets employing three-stage protocol developed by our group. These islets could be used as novel in vitro model for screening hypoglycemics/insulin secretagogues, thus reducing animal experimentation for this purpose and for the future human islet transplantation programs to treat diabetes.

In Vivo Evaluation of the Biocompatibility of Surface Modified Hemodialysis Polysulfone Hollow Fibers in Rat

Polysulfone (Psf) hollow fiber membranes (HFMs) have been widely used in blood purification but their biocompatibility remains a concern. To enhance their biocompatibility, Psf/TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate) composite HFMs and 2-methacryloyloxyethyl phosphorylcholine (MPC) coated Psf HFMs have been prepared. They have been evaluated for in vivo biocompatibility and graft acceptance and compared with sham and commercial membranes by intra-peritoneal implantation in rats at day 7 and 21. Normal body weights, tissue formation and angiogenesis indicate acceptance of implants by the animals. Hematological observations show presence of post-surgical stress which subsides over time. Serum biochemistry results reveal normal organ function and elevated liver ALP levels at day 21. Histological studies exhibit fibroblast recruitment cells, angiogenesis and collagen deposition at the implant surface indicating new tissue formation. Immuno-histochemistry studies show non-activation of MHC molecules signifying biocompatibilty. Additionally, Psf/TPGS exhibit most favorable tissue response as compared with other HFMs making them the material of choice for HFM preparation for hemodialysis applications.

Artificial Bone via Bone Tissue Engineering: Current Scenario and Challenges

Bone provides mechanical support, and flexibility to the body as a structural frame work along with mineral storage, homeostasis, and blood pH regulation. The repair and/or replacement of injured or defective bone with healthy bone or bone substitute is a critical problem in orthopedic treatment. Recent advances in tissue engineering have shown promising results in developing bone material capable of substituting the conventional autogenic or allogenic bone transplants. In the present review, we have discussed natural and synthetic scaffold materials such as metal and metal alloys, ceramics, polymers, etc. which are widely being used along with their cellular counterparts such as stem cells in bone tissue engineering with their pros and cons.