Pawan Kumar Gupta

A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia

BackgroundPeripheral vascular disease of the lower extremities comprises a clinical spectrum that extends from no symptoms to presentation with critical limb ischemia (CLI). Bone marrow derived Mesenchymal Stem Cells (BM- MSCs) may ameliorate the consequences of CLI due to their combinatorial potential for inducing angiogenesis and immunomodulatory environment in situ. The primary objective was to determine the safety of BM- MSCs in patients with CLI.MethodsProspective, double blind randomized placebo controlled multi-center study was conducted in patients with established CLI as per Rutherford classification in category II-4, III-5, or III-6 with infra-inguinal arterial occlusive disease and were not suitable for or had failed revascularization treatment. The primary end point was incidence of treatment – related adverse events (AE). Exploratory efficacy end points were improvement in rest pain, increase in Ankle Brachial Pressure Index (ABPI), ankle pressure, healing of ulcers, and amputation rates. Twenty patients (BM-MSC: Placebo = 1:1) were administered with allogeneic BM-MSCs at a dose of 2 million cells/kg or placebo (PlasmaLyte A) at the gastrocnemius muscle of the ischemic limb.ResultsImprovement was observed in the rest pain scores in both the arms. Significant increase in ABPI and ankle pressure was seen in BM-MSC arm compared to the placebo group. Incidence of AEs in the BM-MSC arm was 13 vs. 45 in the placebo arm where as serious adverse events (SAE) were similar in both the arms (5 in BM-MSC and 4 in the placebo group). SAEs resulted in death, infected gangrene, amputations in these patients. It was observed that the SAEs were related to disease progression and not related to stem cells.ConclusionBM-MSCs are safe when injected IM at a dose of 2 million cells/kg body weight. Few efficacy parameters such as ABPI and ankle pressure showed positive trend warranting further studies.Trial registrationNIH website (http://www.clinicaltrials.gov/ct2/show/NCT00883870)

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.

Chitosan enhances mineralization during osteoblast differentiation of human bone marrow‐derived mesenchymal stem cells, by upregulating the associated genes

Objectives:  Chitosan is widely used as a scaffold for bone tissue engineering. However, up‐to‐date, no previous detailed study has been conducted to elucidate any mechanism of osteogenesis by chitosan itself. Here, we have evaluated effects of chitosan‐coated tissue culture plates on adhesion and osteoblast differentiation processes of human mesenchymal stem cells (hMSCs), isolated from adult bone marrow.

A novel tripolymer coating demonstrating the synergistic effect of chitosan, collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells

The biomimetic approach mimicking in vivo micro environment is the key for developing functional tissue engineered constructs. In this study, we used a tripolymer combination consisting of a natural polymer, chitosan and two extracellular matrix components; collagen type 1 and hyaluronic acid to coat tissue culture plate to evaluate their effect on osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs). The polymers were blended at different mixing ratios and the tissue culture plates were coated either by polyblend method or by surface modification method. hMSCs isolated from adult bone marrow were directed to osteoblast differentiation on the coated plates. Our results showed that the tripolymer coating of the tissue culture plate enhanced mineralization as evidenced by calcium quantification exhibiting significantly higher amount of calcium compared to the untreated or individual polymer coated plates. We found that the tripolymer coated plates having a 1:1 mixing ratio of chitosan and collagen type 1, surface modified with hyaluronic acid is an ideal combination to achieve the synergistic effect of these polymers on in vitro osteogenic differentiation of hMSCs. These results thus, establish a novel biomimetic approach of surface modification to enhance osteoblast differentiation and mineralization. Our findings hold great promise in implementing a biomimetic surface coating to improve osteoconductivity of implants and scaffolds for various orthopaedic and bone tissue engineering applications.

Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells

Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell‐based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin‐4‐sulphate, dermatan sulphate, chondroitin‐6‐sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan‐treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin‐4‐sulphate. Osteoblast differentiation‐associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin‐6‐sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering. Copyright

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice.

BACKGROUND AIMS The success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. METHODS We compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. RESULTS SHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3-4 weeks, which persisted for >60 days. CONCLUSIONS Our results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.

Midbrain Cues Dictate Differentiation of Human Dental Pulp Stem Cells Towards Functional Dopaminergic Neurons

Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell‐based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell‐types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell‐type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III, and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron‐specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired‐like homeodomain transcription factor 3 (Pitx3) as revealed by real‐time RT‐PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic‐neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1, and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca2+ influx in the presence of KCl, unlike control DPSCs. ATP‐stimulated Ca2+ influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrate for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell‐type. J. Cell. Physiol. 229: 1369–1377, 2014.

Dental pulp stem cells immobilized in alginate microspheres for applications in bone tissue engineering

AIM To immobilize dental pulp stem cells (DPSC) in alginate microspheres and to determine cell viability, proliferation, stem cell characteristics and osteogenic potential of the immobilized DPSCs. METHODOLOGY Human DPSCs isolated from the dental pulp were immobilized in 1% w/v alginate microspheres. Viability and proliferation of immobilized DPSCs were determined by trypan blue and MTT assay, respectively. Stem cell characteristics of DPSCs post immobilization were verified by labelling the cells with CD73 and CD90. Osteogenic potential of immobilized DPSCs was assessed by the presence of osteocalcin. Alizarin red staining and O-cresolphthalein complexone method confirmed and quantified calcium deposition. A final reverse transcriptase PCR evaluated the expression of osteogenic markers - ALP, Runx-2 and OCN. RESULTS More than 80% of immobilized DPSCs were viable throughout the 3-week study. Proliferation appeared controlled and consistent unlike DPSCs in the control group. Presence of CD73 and CD90 markers confirmed the stem cell nature of immobilized DPSCs. The presence of osteocalcin, an osteoblastic marker, was confirmed in the microspheres on day 21. Mineralization assays showed high calcium deposition indicating elevated osteogenic potential of immobilized DPSCs. Osteogenic genes- ALP, Runx-2 and OCN were also upregulated in immobilized DPSCs. Surprisingly, immobilized DPSCs in the control group cultured in conventional stem cell media showed upregulation of osteogenic genes and expressed osteocalcin. CONCLUSION Dental pulp stem cells immobilized in alginate hydrogels exhibit enhanced osteogenic potential while maintaining high cell viability both of which are fundamental for bone tissue regeneration.

Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal‐derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF‐MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin‐C treatment. Growth pattern, gene profiling (Oct‐4, Nanog, Sox‐2, Rex‐1), cytoskeletal protein expression (vimentin, nestin) and tri‐lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF‐MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA‐4, and negative for CD34, CD45, HLA‐DR cell‐surface markers and were found to exhibit BM‐MSC‐like characteristics. hESC lines co‐cultured with HF‐MSC feeders showed expression of expected pluripotent transcription factors Oct‐4, Nanog, Sox‐2, GDF‐3, Rex‐1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA‐4, TRA‐1‐81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical‐grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications. J. Cell. Biochem. 112: 1353–1363, 2011.

A Simple and economical route to generate functional hepatocyte‐like cells from hESCs and their application in evaluating alcohol induced liver damage

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two‐step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte‐like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT‐PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF‐3β, SOX‐17, DLX‐5, CXCR4) was consistent and in concurrence with the up‐regulation of the markers for hepatic progenitors [alpha‐feto protein (AFP), HNF‐4α, CK‐19, albumin, alpha‐1‐antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan‐2, 3‐dioxygenase (TDO), glucose 6‐phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down‐regulation of extra embryonic endodermal markers (SOX‐7, HNF‐1β, SNAIL‐1, LAMININ‐1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis. J. Cell. Biochem. 113: 19–30, 2012.