Showing papers by "Rama Shanker Verma published in 2016"

A Patient-Inspired Ex Vivo Liver Tissue Engineering Approach with Autologous Mesenchymal Stem Cells and Hepatogenic Serum.

Abstract: Design and development of ex vivo bioengineered liver tissue substitutes intended for subsequent in vivo implantation has been considered therapeutically relevant to treat many liver diseases that require whole-organ replacement on a long-term basis. The present study focus on patient-inspired ex vivo liver tissue engineering strategy to generate hepatocyte-scaffold composite by combining bone marrow mesenchymal stem cells (BMSCs) derived from cardiac failure patients with secondary hyperbilirubinemia as primers of hepatic differentiation and hepatocyte growth factor (HGF)-enriched sera from same individuals as hepatic inducer. A biodegradable and implantable electrospun fibrous mesh of poly-l-lactic acid (PLLA) and gelatin is used as supporting matrix (average fiber diameter = 285 ± 64 nm, porosity = 81 ± 4%, and average pore size = 1.65 ± 0.77 μm). The fibrous mesh supports adhesion, proliferation, and hepatic commitment of patient-derived BMSCs of adequate stemness using HGF-enriched sera generating metabolically competent hepatocyte-like cells, which is comparable to the hepatic induction with defined recombinant growth factor cocktail. The observed results confirm the combinatorial effects of nanofiber topography and biochemical cues in guiding hepatic specification of BMSCs. The fibrous mesh-hepatocyte construct developed in this study using natural growth factors and BMSCs of same individual is promising for future therapeutic applications in treating damaged livers.

Impaired mitophagy in Fanconi anemia is dependent on mitochondrial fission

Abstract: Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells

Abstract: 3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

Novel TNF-related Apoptotic-inducing Ligand-based Immunotoxin for Therapeutic Targeting of CD25 Positive Leukemia

Abstract: Human TNF-related apoptotic-inducing ligand (TRAIL) has been used successfully for targeted therapy of almost all cancers. Leukemia is the most common type of cancer in children, and despite the advances in therapeutic strategies, the survival rate in leukemia cases is very low. Overexpression of interleukin 2 receptor (IL2R) in hematological malignancies has been utilized to target leukemia. Here, we report an immunotoxin fusion construct of human IL2α and TRAIL for targeting leukemia. Our aim was to develop an immunotoxin to target CD25+ leukemic cells. Recombinant fusion construct comprising human IL2α and TRAIL114–281 was cloned, expressed and purified. Surface expression levels of IL2α and TRAIL receptors (CD25 and DR5 respectively) were compared in four leukemic cell lines and patient-derived peripheral blood mononuclear cells (PBMCs). Efficacy of immunotoxins was tested in cell lines and PBMCs by cell viability assay and compared with receptor expression. The efficacy of IL2-TRAIL was higher than TRAIL alone and showed an IC50 ranging from 0.2-0.8 μM in cell lines. IL2-TRAIL induced cell death in PBMCs from leukemic patients in vitro, which was proportional to CD25 expression. Out of 34 leukemic samples, 24 samples were susceptible to immunotoxin-mediated cytotoxicity. The efficacy of IL2-TRAIL (87.5 %) was significantly high compared to TRAIL protein (29 %) in both myeloid and lymphoid leukemic patient samples. IL2-TRAIL fusion protein was highly specific for CD25+ leukemia and showed 100 % efficacy in lymphocytic leukemia [acute lymphoblastic leukemia and chronic lymphocytic leukemia] that overexpressed CD25.

Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins

Abstract: Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

Evolving Concepts for Use of Stem Cells and Tissue Engineering for Cardiac Regeneration

Abstract: The incidence of cardiovascular disease (CVD) in adults are increasing worldwide with impaired repair mechanisms, leading to tissue and organ failure. With the current advancements, life expectancy has improved and has led to search for new treatment strategies that improves tissue regeneration. Recently, stem cell therapy and tissue engineering has captured the attention of clinicians, scientists, and patients as alternative treatment options. The overall clinical experience of these suggests that they can be safely used in the right clinical setting. Ultimately, large outcome trials will have to be conducted to assess their efficacy. Clinical trials have to be carefully designed and patient safety must remain the key concern. At the same time, continued basic research is required to understand the underlying mechanism of cell-based therapies and cell tissue interactions. This chapter reviews the evolving paradigm of stem cell therapy and tissue engineering approaches for clinical application and explores its implications.

Breaking dogma for future therapy using stem cell - Where we have reached?

Abstract: Stem cells (SCs) are functionally immature cells which have the potential to become any cell type upon stimulation. Stem cells have the unique capacity to self-renew, hence are the most promising cell source for tissue regeneration, treating cellular disorders like Parkinson's disease, to replace dead or dysfunctional cells in various traumas. Stem cells are broadly classified into embryonic stem cells (ESC), adult stem cells (ASC), based on their source and induced pluripotent stem cells (iPSC) which are genetically induced by incorporating several nuclear factors such as Sox2, c-myc, Oct4 and nanog, etc1. Pluripotent stem cells are widely used as model system to study embryonic development and cellular differentiation. Stem cells are found in many adult tissues such as epidermis, ocular, muscle, intestine, bone marrow, brain, adipose etc. including insulin-producing beta cells2,3. Growth factors and small molecules control the signals that drive these cells along the different pathways to produce mature cells from stem or progenitor cells. It is the pioneering basic research on the discovery of these signaling pathways for endoderm and pancreatic cells in early development that has paved the way for making laboratory grown beta cells3. A remarkable progress has been made during the past decade specifically, generating insulin producing cells. These cells represent putative beta cells, shown by expression of transcription factors known to control maturation of beta cells that secrete glucose and secretagogue-induced insulin thus restoring normal blood sugar levels after transplantation in diabetic mice4. Two studies4,5 have reported generating insulin-producing cells that resemble normal beta cells from human pluripotent stem cells that share significant functional features with normal human beta cells. This provides a step forward for a potential cell therapy treatment for diabetes. Photoreceptor loss may cause irreversible damage causing blindness in several retinal diseases. Brain- and retina-derived stem cells when transplanted into adult retina were not integrated into the outer nuclear layer and differentiated into new photoreceptors6. The integration of transplanted cells in the retinal region is controlled by ontogentic stage of the cell. Knowledge of the factors which define the differentiation and integration of the precursor cells in the host tissue would facilitate in identifying and enriching the cells suitable for enhancing function in the damaged region6. In the area of dedifferentiation of stem cells, both neural crest stem cells and mesenchymal stem cells (MSCs) from bone marrow have shown some advantage in cellular therapies to replace neurons in various neurological diseases. Neurons derived from hESCs integrate efficiently into brain circuits in vivo7. Blood vessel derived growth factors stimulate neural stem cells to differentiate neurons and this aids the brain repair itself after injury or disease, as in cases of stroke, traumatic brain injury and dementia8. Neuron formation is highly focused option owing to the multiple diseases includes Alzheimer's disease (AD), cerebral palsy, etc. Stem cells can be used as a vehicle to secrete neurotrophins, which are reduced in patients with AD. However, neuronal regeneration is very limited due to the decreased neurogenesis. Embryonic and iPSCs have been successfully used for neuron based regenerative diseases. Recent studies have shown that MSCs from bone marrow do not migrate or differentiate in the MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) treated striatum9. These factors create neuron differentiation a lucrative research option. Vierbuchen et al10 have shown induction of neuronal differentiation in mouse embryonic and postnatal fibroblasts using factors Ascl1, Brn2, and Myt1l. Caiazzo et al11, described a way to reprogram fibroblasts from mouse and human origin into dopaminergic neurons, without going through a pluripotent stage with intact dopaminergic activity. The in vitro differentiation and functionality of neuron differentiation are being studied, but the in vivo usage and therapeutic application are way from seen11. The mechanism of conversion of fibroblasts into neurons is not well studied, Mash1 gene has been shown to play a major role in the differentiation, and initiating immature neuron formation12. Cells of the neural crest origin have been shown to differentiate into neurons and are an easily available source compared to embryonic and mesenchymal lineage. The hair follicles have been shown to harbour pluripotent neural crest stem cells, and these can be differentiated into melanocyte, neuronal cells, adipose cells and other lineages13. Recent studies achieved a step forward in the development of cell-based therapies in other areas such as deafness. In one such study, cells from human embryonic and foetal stem cells identified as a candidate source have been shown to differentiate into auditory neurons that improve auditory-evoked response thresholds14. Similarly, pancreatic progenitor cells derived from hESCs, hold a promising new treatment for diabetes. Bone marrow mesenchymal and haematopoietic stem cells are being studied to develop better repair strategies for the osteoarticular system and blood disorders like thalassaemia. MSCs derived from human adipose tissues engineered to express suicide gene cytosine deaminase :: uracil phosphoribosyltransferase were used as vehicles to treat against gliobastoma cells15. In context to the current work in the issue, Kumar et al16 found stem cell like cells from human skin and hair follicles were characterized for their differentiation potential into melanocytes and neurons. The study focused on the differentiation potential of stem cells from two different sources into melanocytes and neurons. It revealed the enhanced differentiation ability into melanocytes and neurons, which is very promising for the use of candidate cell type in the area of skin regeneration (melanocytes) and also neuron degenerative diseases (neuron). Several research groups have demonstrated the versatility of embryonic stem cells, which can be differentiated into different cell types. Stem cells have the most possible therapeutic use currently in skin regeneration. The source of cells for the purpose revolves around skin graft cells and follicle cells, which are part of the neural crest, that differentiate into the different cell types of the skin, including melanocyte, keratinocyte and skin progenitor cells, etc. Hair follicle is a better source of stem cells for skin regeneration therapy since it has enhanced potential to differentiate into melanocyte17. The epidermis houses skin stem cells owing to the excessive wear and tear experienced by the organ, which help in skin repair. Skin stem cells comprise epidermal stem cells, hair follicle stem cells and melanocyte stem cells. O’ Connor et al18 have shown that the epidermal stem cells can be utilized to form skin grafts for the burn patients. Future direction in this area can include better skin regeneration and that should include rapid proliferating cells with maintenance of stemness, along with regeneration of nerves. Improved culture conditions include supplementing growth factors which induce cell type specific transcription factors that can yield enhanced potential cell type to differentiate into any cell types of skin origin. The formation of neuronal cells in the skin graft might increase the sensation in the regenerated area.