Dr. Ajay Kumar is currently working as a Postdoctoral Researcher at the Department of Ophthalmology, School of Medicine, Stanford University. Dr. Kumar is a stem cell biologist by training and has been working in the stem cell field for more than a decade. His primary interest is to devise novel stem cell-based and cell-free therapies for the treatment of glaucoma, other neurodegenerative diseases, and aging. He devised a new method to mimic the development of the human retina in a dish and a new treatment strategy for glaucoma using stem cell secretome. Dr. Kumar is the recipient of multiple awards and has published more than 25 peer-reviewed papers in esteemed journals. He wishes to establish regenerative medicine-based therapies for different diseases which can benefit patients.
Honors & Awards
SPARK Translational Research Program Pilot Grant, Stanford University (January 2022)
ISER/BrightFocus Travel award, Bright Focus Foundation, USA (Aug 2021)
Amir Chand Gold medal, PGIMER, Chandigarh, India (2021)
Weigand Fellowship in Regenerative Ophthalmology, University of Pittsburgh, USA (2018-19)
Amir Chand Silver medal, PGIMER, Chandigarh, India (2018)
R Srinivasan award, Indian Biophysical Society (2017)
Young Innovator Award in medicine, ABMS, PGIMER, Chandigarh, India (2016)
First Rank PhD Entrance Exam, PGIMER, Chandigarh, India (2012)
Research Fellowship, CSIR, India (2010-2015)
First Rank MSc Entrance Exam, Kurukshetra University, India (2007)
SPARK at Stanford
Master of Science, Kurukshetra University (2009)
Bachelor of Science, Kurukshetra University (2007)
Doctor of Philosophy, Postgraduate Institute of Medical Education (2017)
Postdoc, Stanford University, Designing therapy for optic neuropathy (2021)
Postdoc, University of Pittsburgh, Treatment of vision loss using stem cell-based and cell-free therapy (2021)
PhD, PGIMER, Chandigarh, India, Stem Cell Biology (2017)
Yaping Liao, Postdoctoral Faculty Sponsor
Yiqin Du, Ajay Kumar. "United States Patent WO2021178977 COMPOSITIONS AND METHODS FOR TREATING OCULAR DISORDERS", University of Pittsburgh, Sep 10, 2021
Current Research and Scholarly Interests
My main project is based on devising a treatment for Anterior ischemic optic neuropathy (AION) by using skin biopsies from AION patients and differentiating fibroblast to retinal ganglion cells ultimately. I am also working on strategies to regenerate retinal ganglion cells in optic nerve crush model.
Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.
Investigative ophthalmology & visual science
2022; 63 (2): 12
Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.
View details for DOI 10.1167/iovs.63.2.12
View details for PubMedID 35129590
Regenerative therapy for the Cornea.
Progress in retinal and eye research
The cornea is the outmost layer of the eye, unique in its transparency and strength. The cornea not only transmits the light essential for vision, also refracts light, giving focus to images. Each of the three layers of the cornea has properties essential for the function of vision. Although the epithelium can often recover from injury quickly by cell division, loss of limbal stem cells can cause severe corneal surface abnormalities leading to corneal blindness. Disruption of the stromal extracellular matrix and loss of cells determining this structure, the keratocytes, leads to corneal opacity. Corneal endothelium is the inner part of the cornea without self-renewal capacity. It is very important to maintain corneal dehydration and transparency. Permanent damage to the corneal stroma or endothelium can be effectively treated by corneal transplantation; however, there are drawbacks to this procedure, including a shortage of donors, the need for continuing treatment to prevent rejection, and limits to the survival of the graft, averaging 10-20 years. There exists a need for new strategies to promote regeneration of the stromal structure and restore vision. This review highlights critical contributions in regenerative medicine with the aim of corneal reconstruction after injury or disease. These approaches include corneal stromal stem cells, corneal limbal stem cells, embryonic stem cells, and other adult stem cells, as well as induced pluripotent stem cells. Stem cell-derived trophic factors in the forms of secretomes or exosomes for corneal regeneration are also discussed. Corneal sensory nerve regeneration promoting corneal transparency is discussed. This article provides description of the up-to-date options for corneal regeneration and presents exciting possible avenues for future studies toward clinical applications for corneal regeneration.
View details for DOI 10.1016/j.preteyeres.2021.101011
View details for PubMedID 34530154
Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma.
2021; 11 (9)
Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm's canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.
View details for DOI 10.3390/biom11091258
View details for PubMedID 34572471
- Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model (vol 6, 216, 2018) COMMUNICATIONS BIOLOGY 2021; 4 (1): 456
Stem cell transplantation rescued a primary open-angle glaucoma mouse model
Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if transplanted stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.
View details for DOI 10.7554/eLife.63677
View details for Web of Science ID 000618527700001
View details for PubMedID 33506763
View details for PubMedCentralID PMC7864631
Stem cell-free therapy for glaucoma to preserve vision
View details for DOI 10.1101/2021.06.18.449038
Two-step induction of trabecular meshwork cells from induced pluripotent stem cells for glaucoma
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2020; 529 (2): 411-417
Glaucoma is a leading cause of irreversible blindness worldwide. Reducing intraocular pressure is currently the only effective treatment. Elevated intraocular pressure is associated with increased resistance of the outflow pathway, mainly the trabecular meshwork (TM). Despite great progress in the field, the development of novel and effective treatment for glaucoma is still challenging. In this study, we reported that human induced pluripotent stem cells (iPSCs) can be cultured as colonies and monolayer cells expressing OCT4, alkaline phosphatase, SSEA4 and SSEA1. After induction to neural crest cells (NCCs) positive to NGFR and HNK1, the iPSCs can differentiate into TM cells. The induced iPSC-TM cells expressed TM cell marker CHI3L1, were responsive to dexamethasone treatment with increased expression of myocilin, ANGPTL7, and formed CLANs, comparable to primary TM cells. To the best of our knowledge, this is the first study that induces iPSCs to TM cells through a middle neural crest stage, which ensures a stable NCC pool and ensures the high output of the same TM cells. This system can be used to develop personalized treatments using patient-derived iPSCs, explore high throughput screening of new drugs focusing on TM response for controlling intraocular pressure, and investigate stem cell-based therapy for TM regeneration.
View details for DOI 10.1016/j.bbrc.2020.05.225
View details for Web of Science ID 000551875700045
View details for PubMedID 32703444
View details for PubMedCentralID PMC7385283
Assessment of Post-thaw Quality of Dental Mesenchymal Stromal Cells After Long-Term Cryopreservation by Uncontrolled Freezing
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
2020; 191 (2): 728-743
Cryopreservation abilities of dental tissue-derived mesenchymal stromal cells (DMSCs) including dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSC) play an important role in the applications of these cells in clinical settings. In this context, we checked whether storage at - 80 °C in 10% DMSO for a longer period has any adverse effect on the functionality and genetic stability. We carried our studies on DPSC and DFSC samples that were revived after a maximum of 5 years of cryopreservation. We observed that even after long-term uncontrolled freezing at - 80 °C, these cells survived and proliferated efficiently. The assessment was made based on their post-thaw morphology, immunophenotypes, differentiation potential, growth kinetics, and genetic features. These cells retained the expression of stemness markers, differentiation ability and maintained their normal karyotype. Our results indicated no significant morphological or immunophenotypic differences between the cryopreserved DMSCs and the fresh DMSCs. Our study implies that mesenchymal stromal cells derived from the dental tissue origin are very robust and do not require any sophisticated preservation protocols. Thus, these can be an ideal source for research, stem cell banking, as well as successful clinical applications in tissue engineering and cell-based therapeutics. Graphical Abstract Schematic diagram showing the cryopreservation of DMSCs by uncontrolled freezing at -80 c has no adverse effects on their functionality and genetic stability.
View details for DOI 10.1007/s12010-019-03216-6
View details for Web of Science ID 000541559900019
View details for PubMedID 31853872
Differentiation of Trabecular Meshwork Stem Cells into Retinal Ganglion Cells
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
View details for Web of Science ID 000554495705100
BRAF V600E mutation in childhood Langerhans cell histiocytosis correlates with multisystem disease and poor survival
BLOOD CELLS MOLECULES AND DISEASES
2020; 82: 102356
Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia of children with systemic involvement and poor outcome. The altered RAS-RAF-MEK-ERK cell signalling pathway due to somatic mutation of BRAF V600E is the most common genetic abnormality associated with the disease. In the current study, we highlight the frequency of BRAF V600E in our cohort of LCH cases (n = 31) and its relation with clinical outcome. On Real-Time PCR and Sanger sequencing, BRAF V600E was detected in 6/31 (19%) patients. All cases positive for BRAF V600E mutation had multisystem involvement/disseminated disease compared to BRAF mutation negative cases (100% v/s 41%, p = 0.0348). Univariate analysis also revealed significant correlation of mutation positivity with risk category (p = 0.09). The event free survival and overall survival at 36 months for BRAF mutation positive group compared to mutation negative group was 17% v/s 72% (Log rank test p = 0.0110) and 32.5% v/s 82% (p = 0.0330), respectively. In our study, BRAF V600E positivity was low (19%) however, all positive cases had multisystem involvement and a poor three year survival confirming BRAF V600E to be a poor prognostic marker.
View details for DOI 10.1016/j.bcmd.2019.102356
View details for Web of Science ID 000521506900002
View details for PubMedID 32171112
α5β1 Integrin Promotes Anchoring and Integration of Transplanted Stem Cells to the Trabecular Meshwork in the Eye for Regeneration.
Stem cells and development
2020; 29 (5): 290-300
Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5β1 integrin antibody. TMSCs and TMSCs with α5β1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5β1 integrin than fibroblasts, but similar levels of αvβ3 and αvβ5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5β1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5β1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5β1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5β1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.
View details for DOI 10.1089/scd.2019.0254
View details for PubMedID 31854234
View details for PubMedCentralID PMC7047116
Stem Cells from Human Trabecular Meshwork Hold the Potential to Develop into Ocular and Non-Ocular Lineages After Long-Term Storage
STEM CELLS AND DEVELOPMENT
2020; 29 (1): 49-61
Stem cells from the eye hold a great potential for vision restoration and can also be used for regeneration in other tissues. In this study, we characterized the stem cell properties of Trabecular meshwork stem cells (TMSCs) after long-term cryopreservation (∼8 years). TMSCs derived from four donors were examined for their viability and proliferation, as well as stem cell marker expression. Spheroid formation, colony formation, and multipotency were investigated. We observed that TMSCs were fully viable with variable proliferation ability. They expressed the stem cell markers CD90, CD166, CD105, CD73, OCT4, SSEA4, Notch1, KLF4, ABCG2, Nestin, and HNK1 detected by flow cytometry, quantitative polymerase chain reaction, or immunofluorescent staining. They could form spheroids and colonies after thawing. All TMSCs were able to differentiate into osteocytes, neural cells, and trabecular meshwork (TM) cells, but not adipocytes. Differentiated TM cells responded to dexamethasone treatment with increased expression of myocilin and angiopoietin-like 7 (ANGPTL7). In a nutshell, our study demonstrated that TMSCs retain their stem cell properties after long-term cryopreservation and hence can be an effective cell therapy source for various clinical applications.
View details for DOI 10.1089/scd.2019.0169
View details for Web of Science ID 000505317100005
View details for PubMedID 31680626
View details for PubMedCentralID PMC6931915
Human Embryonic Stem Cells Differentiate into Trabecular Meshwork Cells
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
View details for Web of Science ID 000488800704102
Audit of Quality and Quantity of Nucleic Acid Yield from Pediatric Acute Leukemia Cases Following a Bio-banking Initiative
INDIAN JOURNAL OF HEMATOLOGY AND BLOOD TRANSFUSION
2019; 35 (1): 77-82
Information which can be harvested from a biological sample has greatly improved with advancements in diagnostic technologies. However, in developing countries, the awareness about usefulness of bio-banking concept is lacking and centres which do offer it, depend mainly on - 20 or - 80 °C for sample storage due to lack of sophisticated infrastructure like vapour phase nitrogen storage preservation. Hence in these resource constraint settings, timely audit of quality of nucleic acids extractable from samples stored is of utmost importance. In this study, we explore the effect of - 20 °C storage over nucleic acids (DNA/RNA) isolated from blood samples of 180 patients with various leukaemia's following a bio-banking initiative. We observed that the integrity and quality of both DNA and RNA were maintained in 70 and 80% samples respectively over time as reflected by their concentration measurements and inherent uniform expression of housekeeping gene GAPDH. Only 3.7% of the RNA samples and 4.2% of the DNA samples yielded very low concentrations despite minimizing processing and technical loss. In nutshell, audit of our biobank sample yield highlights that storage of blood samples at - 20 °C does not compromise the fidelity of nucleic acids for future diagnostic and research work in a resource constraint setting.
View details for DOI 10.1007/s12288-018-0975-4
View details for Web of Science ID 000458245100014
View details for PubMedID 30828152
View details for PubMedCentralID PMC6369071
Secretome proteins regulate comparative osteogenic and adipogenic potential in bone marrow and dental stem cells
2018; 155: 129-139
Dental stem cells (DMSC) have been studied extensively since their early discovery. However, the data regarding osteogenic potential of DMSC with other cell types is sparse and the secretome proteins underlying these differences have not been explored. In this study, we have compared the osteogenic and adipogenic potential of DMSC with Bone Marrow Stem cells (BMSC) and reported the contribution of secretome proteins in controlling their differentiation.Osteogenic potential of these stem cells was compared by mineralization assay, alkaline phosphatase (ALP) assay, immunofluorescence of dentine sialo phosphoprotein (DSPP) & qPCR for osteogenic genes. Adipogenic potential was compared by Oil Red O staining and qPCR for PPAR-γ, leptin & adipsin. Proteomic analysis of secretome was performed by employing WATERS nano Lc-MS/MS system.We observed a higher osteogenic potential in DMSC, especially dental pulp stem cells (DPSC) as compared to BMSC population but adipogenic potential was found to be better in BMSC as compared to DMSC. Deeper investigations into secretome of these cells by Lc-MS/MS revealed the presence of proteins pertaining to osteogenic and adipogenic lineage. Presence of some important proteins regulating osteogenic (DSPP, BMP7, DDR2, USP9X) and adipogenic differentiation (NCOA2, PEG10, LPA) in secretome of BMSC and DMSC reflected the role of paracrine factors during differentiation.Our study provides first evidence regarding regulation of osteogenic/adipogenic potential by secretome proteins in DMSC and BMSC. DMSC especially DPSC and its secretome show an inherent tendency for higher osteogenic differentiation and lower adipogenic differentiation, these may be potential candidates for effective future therapy in osteoporosis where disturbance of osteocyte/adipocyte homeostasis is reported.
View details for DOI 10.1016/j.biochi.2018.10.014
View details for Web of Science ID 000450903000015
View details for PubMedID 30367923
Stemness and Regenerative Potential of Corneal Stromal Stem Cells and Their Secretome After Long-Term Storage: Implications for Ocular Regeneration
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2018; 59 (8): 3728-3738
To assess the stemness and regenerative potential of cryopreserved corneal stromal stem cells (cryo-CSSCs) after long-term storage. We also used the secretome from these cells to observe the effect on wound-healing capacity of corneal fibroblasts and on the expression of fibrotic markers during wound healing.CSSCs were obtained from three donors and stored in liquid nitrogen for approximately 10 years. Post thaw, cryo-CSSCs were characterized for stemness using phenotypic and genotypic markers along with colony-forming efficiency and three-dimensional spheroid formation. Multilineage differentiation was observed by differentiation into osteocytes, adipocytes, neural cells, and keratocytes. Secretome was harvested by culturing cryo-CSSCs in log phase. Wound-healing capacity was observed by live-cell time-lapse microscopy. Statistical analysis was done using 1-way ANOVA and Tukey posttest.CSSCs displayed good viability post thaw and showed >90% expression of stem cell markers CD90, CD73, CD105, STRO1, and CD166. cryo-CSSCs also expressed stem cell genes OCT4, KLF4, and ABCG2, and could also form colonies and three-dimensional spheroids. Multipotency assessment showed that all three cryo-CSSCs could differentiate into osteocytes, adipocytes, neural cells, as shown by β-III tubulin and neurofilament antibody staining and corneal keratocytes as observed by staining for Kera C, J19, and collagen V antibodies. The secretome derived from these three populations could promote the wound healing of corneal fibroblasts and reduce the expression of fibrotic markers SPARC and fibronectin.CSSCs maintained their stemness and multipotency after long-term storage, and secretome derived from these cells can be of paramount importance for corneal regeneration and prevention of fibrosis.
View details for DOI 10.1167/iovs.18-23824
View details for Web of Science ID 000440245200002
View details for PubMedID 30046814
View details for PubMedCentralID PMC6059729
Stemness and regenerative effects of trabecular meshwork stem cells/secretome after long-term storage
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442932804206
Possible Autologous Stem Cell Resources for Trabecular Meshwork Regeneration
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442932804207
Epithelial to mesenchymal transition induces stem cell like phenotype in renal cell carcinoma cells
CANCER CELL INTERNATIONAL
2018; 18: 57
Metastatic dissemination of solid tumors is often initiated by reactivation of an embryonic development program, epithelial-to-mesenchymal-transition (EMT). EMT has been associated with acquiring invasiveness and resistance to conventional therapies. However, the precise role of EMT during renal cell carcinoma is still debatable and is under investigation. In this context, our study is designed to evaluate the role of cyclosporine (CsA) and transforming growth factor-β (TGFβ) administration in inducing EMT-like state in renal carcinoma cells. We also studied the associated phenotypic changes which may lead to tumor metastasis.The morphological changes in renal cell carcinoma cells (A498) treated with TGF-β/CsA were observed by microscopy. Atomic force microscope was used to evaluate the changes in elasticity of cells treated with TGF-β/CsA. The expression of mesenchymal and chemoresistance genes were checked by RT-PCR. Assays for migration, invasion, sphere formation ability and expression of cancer stem cell-like phenotypes were done to evaluate the metastatic potential of these cells. Lineage specific differentiations were also done to determine the acquisition of stem-cell like phenotype.Our results showed that treatment with TGF-β/CsA led to loss of epithelial characteristics and gain of mesenchymal phenotype in vitro. Changes in shape and elastic properties of the cancer cells favoured metastatic progression, increased tumorisphere formation and invasiveness post treatment. We also observed higher expression of chemoresistance and stemness markers in EMT-induced cells. These cells also differentiated to various lineages like osteoblasts, adipocytes, neural and hepatic cells when induced with the respective differentiation media.We concluded that TGF-β/CsA treatment led to acquisition of EMT-like cancer stem cells phenotype that enhanced local invasion and dissemination of renal carcinoma cells. This subpopulation of cells with EMT-like phenotype a can provide a better perception of the metastatic process. This can provide an in vitro system for testing pharmaceuticals for modulating EMT as a viable strategy within the therapeutic armamentarium for RCC patients. The results of our findings also suggest that CsA directly induced EMT like changes in epithelial cell which may be responsible for the potential risk of malignancy in transplant patients.
View details for DOI 10.1186/s12935-018-0555-6
View details for Web of Science ID 000429714800001
View details for PubMedID 29681769
View details for PubMedCentralID PMC5896088
Insights into cell-free therapeutic approach: Role of stem cell "soup-ernatant".
Biotechnology and applied biochemistry
2018; 65 (2): 104-118
Current advances in medicine have revolutionized the field of regenerative medicine dramatically with newly evolved therapies for repair or replacement of degenerating or injured tissues. Stem cells (SCs) can be harvested from different sources for clinical therapeutics, which include fetal tissues, umbilical cord blood, embryos, and adult tissues. SCs can be isolated and differentiated into desired lineages for tissue regeneration and cell replacement therapy. However, several loopholes need to be addressed properly before this can be extended for large-scale therapeutic application. These include a careful approach for patient safety during SC treatments and tolerance of recipients. SC treatments are associated with a number of risk factors and require successful integration and survival of transplanted cells in the desired microenvironment with concurrent tissue regeneration. Recent studies have focused on developing alternatives that can replace the cell-based therapy using paracrine factors. The development of stem "cell free" therapies can be devoted mainly to the use of soluble factors (secretome), extracellular vesicles, and mitochondrial transfer. The present review emphasizes on the paradigms related to the use of SC-based therapeutics and the potential applications of a cell-free approach as an alternative to cell-based therapy in the area of regenerative medicine.
View details for DOI 10.1002/bab.1561
View details for PubMedID 28321921
Molecular spectrum of secretome regulates the relative hepatogenic potential of mesenchymal stem cells from bone marrow and dental tissue
2017; 7: 15015
Liver regeneration is a spontaneous process that occurs after liver injury, but acute liver failure is a complex and fatal disease which is difficult to treat. Cell-based therapies are promising alternative therapeutic approach for liver failure and different cell sources have been tested in this regard. We investigated the comparative hepatogenic potential of human bone marrow stem cells (BMSC) with stem cells derived from human dental pulp (DPSC), apical papilla (SCAP) and follicle (DFSC) during this study. Hepatogenic potential of stem cells was assessed by functional assays at both genetic and protein level. We observed higher expression of most of the hepatic markers post differentiation in DPSCs compared to other cell types. LC-MS/MS analysis of stem cell secretome revealed the presence of different proteins related to hepatogenic lineage like growth arrest specific protein 6, oncostatin M, hepatocyte growth factor receptor etc. Interactome and Reactome pathway analysis revealed the interaction of DPSC/SCAP secretome proteins and these proteins were found to be associated with various pathways involved in lipid transport and metabolism. To the best of our knowledge, this is the first study regarding detailed investigation of hepatogenic potential of BMSCs v/s DMSCs (DPSC, SCAP & DFSC) along-with secretome characterization.
View details for DOI 10.1038/s41598-017-14358-0
View details for Web of Science ID 000414644900002
View details for PubMedID 29118330
View details for PubMedCentralID PMC5678086
Secretome Cues Modulate the Neurogenic Potential of Bone Marrow and Dental Stem Cells
2017; 54 (6): 4672-4682
Dental tissue is emerging as a promising source of stem cells especially in nerve regeneration mainly due to their neural origin and ease of harvest. We isolated dental stem cells from three sources, namely, dental pulp (DPSCs), dental follicle (DFSCs), and apical papilla (SCAP), and explored the efficacy of each towards neural differentiation in comparison to bone marrow-derived stem cells. The neural differentiation potential was assessed by expression of various neural markers and neurosphere assay. We observed that DPSCs were inherently predisposed towards neural lineage. To further delineate the paracrine cues responsible for the differences in neural differentiation potential, we harvested the conditioned secretome from each of the stem cell population and observed their effect on colony formation, neurite extension, and neural gene expression of IMR-32, a pre-neuroblastic cell line. We found that neural differentiation was significantly enhanced when IMR-32 cells were treated with secretome derived from DMSCs as compared to the same from BMSCs. Th1/Th2/Th17 cytokine array revealed DPSC secretome had higher expression of the cytokines like GCSF, IFNγ, and TGFβ that promote neural differentiation. Thus, we concluded that DPSCs may be the preferred source of cells for obtaining neural lineage among the four sources of stem cells. Our results also indicate that the DPSC-secreted factors may be responsible for their propensity towards neural differentiation. This study suggests that DPSCs and their secretomes can be a potentially lucrative source for cell-based and "cell-free" (secretome) therapy for neural disorders and injury.
View details for DOI 10.1007/s12035-016-0011-3
View details for Web of Science ID 000405710900060
View details for PubMedID 27422132
Emergence through delicate balance between the steric factor and molecular orientation: a highly bright and photostable DNA marker for real-time monitoring of cell growth dynamics
2017; 53 (17): 2571-2574
Due to its remarkable structural and optical stability in biological milieu, a newly developed small molecule based DNA marker with high brightness (Φ = 0.51, brightness = 26 300 M-1 cm-1) and almost zero toxicity has been explored. For the first time to our knowledge, this approach has provided real-time monitoring of growth and reproductive potency of living cells beyond nine days.
View details for DOI 10.1039/c6cc09355h
View details for Web of Science ID 000395873100009
View details for PubMedID 28191554
Quinazoline derivatives as cathepsins B, H and L inhibitors and cell proliferating agents
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
2017; 94: 719-727
Cysteine Cathepsins well known to be involved in cancer, inflammation and regulation of degenerative processes like apoptosis have become specific targets in drug designing. The potential of quinazolines and their derivatives in medicinal chemistry led us to synthesise a novel series of seven compounds of quinazolines to evaluate their effect on cathepsins and cellular aspects of HepG2 cells. In the present work we report the solvent free microwave assisted synthesis of (E)-8-benzylidene-5,6,7,8-tetrahydro-2,4-diarylquinazolines as inhibitors of mammalian hepatic cysteine proteases viz. Cathepsins B, H and L. In vitro inhibition of Cathepsins B, H and L is correlated well with in vitro studies when tested using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay on HepG2 cells, hepatocellular carcinoma cell line. The studies have been extended to evaluate the type of inhibition exhibited by the individual enzyme. Out of the seven compounds 1g i.e. (E)-8-(4-fluorobenzylidene)-4-(4-fluorophenyl)-2-phenyl-5, 6, 7, 8-tetrahydroquinazoline has been found to be most inhibitory for Cathepsins B, H and L to a maximum extent with the Ki values of 10-10M, 10-10M and 10-9M order respectively. In silico studies of all compounds have also been done at the active sites of Cathepsin B, H and L.
View details for DOI 10.1016/j.ijbiomac.2016.10.001
View details for Web of Science ID 000390621900080
View details for PubMedID 27780761
Selenium Incorporated Cationic Organochalcogen: Live Cell Compatible and Highly Photostable Molecular Stain for Imaging and Localization of Intracellular DNA
ACS APPLIED MATERIALS & INTERFACES
2016; 8 (17): 10690-10699
Successful integration of selenium unit into a newly designed cationic chemical architecture led to the development of a highly photostable molecular maker PA5 to be used in fluorescence microscopy as cellular nucleus staining agent for longer duration imaging under continuous laser illumination. Adaptation of a targeted single-atom modification strategy led to the development of a series of proficient DNA light-up probes (PA1-PA5). Further, their comparative photophysical studies in the presence of DNA revealed the potential of electron rich heteroatoms of chalcogen family in improving binding efficiency and specificity of molecular probes toward DNA. The findings of cell studies confirmed the outstanding cell compatibility of probe PA5 in terms of cell permeability, biostability, and extremely low cytotoxicity. Moreover, the photostability experiment employing continuous laser illumination in solution phase as well as in cell assay (both fixed and live cells) revealed the admirable photobleaching resistance of PA5. Finally, while investigating the phototoxicity of PA5, the probe was found not to exhibit light-induced toxicity even when irradiated for longer duration. All these experimental results demonstrated the promising standing of PA5 as a futuristic cell compatible potential stain for bioimaging and temporal profiling of DNA.
View details for DOI 10.1021/acsami.6b00675
View details for Web of Science ID 000375520700006
View details for PubMedID 27066840
Biomolecular recognition at the cellular level: geometrical and chemical functionality dependence of a low phototoxic cationic probe for DNA imaging
JOURNAL OF MATERIALS CHEMISTRY B
2016; 4 (28): 4895-4900
A combined approach was adopted to understand the impact of structural geometry as well as suitable chemical functionality of a molecular probe on its efficient binding in the minor groove of DNA. The development of a small chemical library of different lutidinium conjugates (P1-P5) and molecular simulations using DFT calculations clearly demonstrated that the semilunar conformation of a molecular probe equipped with requisite chemical functionality is the key parameter for its proper binding in the minor groove of DNA. The comparative optical responses of these probes (P1-P5) coupled with the theoretical studies illustrated that only P3 displayed considerable fluorescence enhancement in the presence of DNA because of its semilunar geometry and special chemical architecture. Furthermore, the bioassays clearly revealed that the probe can penetrate the cell membrane of live as well as dead cells without the help of any permeabilization agent. Microscopic cellular imaging established that probe P3 can stain the nuclear region of the cells with high contrast and negligible cytoplasmic spillage without causing any cellular deterioration. The specificity and binding efficiency of P3 toward DNA were established by performing DNase/RNase digest tests and gel electrophoresis experiments. Most importantly, P3 exhibited minimum phototoxicity and high photobleaching resistance in cellular medium under continuous exposure to a light source, which are highly desirable for real time monitoring of many biological events. Altogether, the investigated properties of P3 shed light on its admirable and persuasive standing as a cell-compatible, bright and photostable molecular probe for nuclear imaging in various bio-medical applications.
View details for DOI 10.1039/c6tb00787b
View details for Web of Science ID 000379570000012
View details for PubMedID 32263148
- Cathepsin B, H and L inhibitors as cell proliferating agents: design, synthesis, computational and pharmacological studies of some novel 2-(2-naphthoyl)-6,6-dimethyl-3-aryl-2,3,6,7-tetrahydrobenzofuran-4(5H)-ones RSC ADVANCES 2016; 6 (41): 34588-34599
Effect of uncontrolled freezing on biological characteristics of human dental pulp stem cells
CELL AND TISSUE BANKING
2015; 16 (4): 513-522
Human dental pulp stem cells (hDPSCs) hold great promise as a source of adult stem cells for utilization in regenerative medicine. Successful storage and post thaw recovery of DPSCs without loss of function is a key issue for future clinical application. Most of the cryopreservation methods use controlled rate freezing and vapor phase nitrogen to store stem cells. But these methods are both expensive and laborious. In this study, we isolated DPSCs from a patient undergoing impacted mandibular third molar extraction. We adopted eight different methods of cryopreservation at -80 °C for long term storage of the DPSC aliquots. Various parameters like proliferation, cell death, cell cycle, retention of stemness markers and differentiation potential were studied post cryopreservation period of 1 year. We observed successful recovery of stem cells in every method and a significant difference in proliferation potential and cell death between samples stored by different methods. However, post thaw, all cells retained their stemness markers. All DPSCs stored by different methods were able to differentiate into osteoblast like cells, adipocytes and neural cells. Based on these parameters we concluded that uncontrolled freezing at a temperature of -80 °C is as effective as controlled freezing using ethanol vessels and other cryopreservation methods. To the best of our knowledge, our study provides the first proof of concept that long term storage in uncontrolled freezing of cells at -80 °C in 10 % DMSO does not affect the revival capacity of hDPSCs. This implies that DPSCs may be used successfully for tissue engineering and cell based therapeutics even after long term, uncontrolled cryopreservation.
View details for DOI 10.1007/s10561-015-9498-5
View details for Web of Science ID 000365868900004
View details for PubMedID 25663639
The voyage of stem cell toward terminal differentiation: a brief overview
ACTA BIOCHIMICA ET BIOPHYSICA SINICA
2012; 44 (6): 463-475
Presently, worldwide attempts are being made to apply stem cells and stem cell-derived products to a wide range of clinical applications and for the development of cell-based therapies. In order to harness stem cells and manipulate them for therapeutic application, it is very important to understand the basic biology of stem cells and identify the factors that govern the dynamics of these cells in the body. Several signaling pathways have emerged as key regulators of stem cells. Some of these signaling pathways regulate the stem cell's proliferative capacity and therefore act as direct regulators of the stem cell, whereas others are involved in shaping and maintaining the stem cell niche and therefore act as indirect regulators of the stem cell. It is difficult to identify which signaling pathways critically affect the stem cell's behavior and which are important for maintaining the quiescent population. A stem cell receives different extrinsic signals compared with the bulk population and responds to them differently. In order to manipulate these adult cells for therapeutic approaches it is crucial to identify how signaling pathways regulate stem cells either directly by regulating proliferative status or indirectly by influencing the niche. The main challenge is to identify whether different factors provide diverse extrinsic signals to the stem cell and its daughter cell population, or whether there are intrinsic differences in stem cell and daughter cell populations that is reflected in their behavior. In this study, we will focus on the various aspects of stem cell biology and differentiation, as well as exploring the potential strategies to intervene the differentiation process in order to obtain the desired yield of cells applicable in regenerative medicine.
View details for DOI 10.1093/abbs/gms027
View details for Web of Science ID 000304830700001
View details for PubMedID 22562866