Publications by authors named "Ramesh R Kaini"

10 Publications

  • Page 1 of 1

Neurotrophic Factors Secreted by Induced Pluripotent Stem Cell-Derived Retinal Progenitors Promote Retinal Survival and Preservation in an Adult Porcine Neuroretina Model.

J Ocul Pharmacol Ther 2021 Mar 3. Epub 2021 Mar 3.

Ocular and Sensory Trauma Task Area, United States Army Institute of Surgical Research, Fort Sam Houston, Texas, USA.

Paracrine factors released by pluripotent stem cells have shown great potential as therapeutic agents in regenerative medicine. The purpose of this study was to characterize trophic factor secretion of retinal progenitor cells (RPCs) derived from human induced pluripotent stem cells (iPSCs) and to assess its impact on retinal survival . RPCs were generated from human 3D1 iPSCs following previously established protocols with modifications. Conditioned medium (CM) was harvested from iPSC-derived retinal progenitors and analyzed for trophic factor composition through multiplex enzyme-linked immunosorbent assay. Retina-preserving capability of the collected CM was examined using a degenerative porcine neuroretina model. Viability of the CM-treated retina explants was evaluated using the resazurin-based PrestoBlue reagent, whereas the lactate dehydrogenase (LDH) assay was used to assess retinal cytotoxicity. Retina explants were also analyzed morphologically through immunohistochemistry for glial cell activation and apoptosis. We have successfully generated and characterized iPSC-derived RPCs that secreted an array of neuroprotective factors, including osteopontin, hepatocyte growth factor, stromal cell-derived factor 1, and insulin-like growth factor-1. Retina explants cultured in CM derived from iPSC-RPCs (iPSC-RPC-CM) showed better preservation of the retinal microarchitecture and fewer terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) nuclei, and reduced reactive gliosis. Furthermore, we saw a reduction in extracellular LDH levels in CM-treated retina explants, which also exhibited higher metabolic activity than the untreated controls. iPSC-derived RPCs secrete many trophic factors that have been shown to promote neuroprotection, tissue repair, and regeneration in the retina. Overall, we have demonstrated the neuroprotective effects of iPSC-RPC-CM through a degenerative neuroretina model .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/jop.2020.0088DOI Listing
March 2021

Utility of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium for an In Vitro Model of Proliferative Vitreoretinopathy.

Adv Exp Med Biol 2019 ;1186:33-53

Ocular Trauma Task Area, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston, TX, USA.

The advent of stem cell technology, including the technology to induce pluripotency in somatic cells, and direct differentiation of stem cells into specific somatic cell types, has created an exciting new field of scientific research. Much of the work with pluripotent stem (PS) cells has been focused on the exploration and exploitation of their potential as cells/tissue replacement therapies for personalized medicine. However, PS and stem cell-derived somatic cells are also proving to be valuable tools to study disease pathology and tissue-specific responses to injury. High-throughput drug screening assays using tissue-specific injury models have the potential to identify specific and effective treatments that will promote wound healing. Retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE) are well characterized cells that exhibit the phenotype and functions of in vivo RPE. In addition to their role as a source of cells to replace damaged or diseased RPE, iPS-RPE provide a robust platform for in vitro drug screening to identify novel therapeutics to promote healing and repair of ocular tissues after injury. Proliferative vitreoretinopathy (PVR) is an abnormal wound healing process that occurs after retinal tears or detachments. In this chapter, the role of iPS-RPE in the development of an in vitro model of PVR is described. Comprehensive analyses of the iPS-RPE response to injury suggests that these cells provide a physiologically relevant tool to investigate the cellular mechanisms of the three phases of PVR pathology: migration, proliferation, and contraction. This in vitro model will provide valuable information regarding cellular wound healing responses specific to RPE and enable the identification of effective therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-030-28471-8_2DOI Listing
October 2019

Polarized Secretion of Matrix Metalloproteinases and Their Inhibitors by Retinal Pigment Epithelium Derived from Induced Pluripotent Stem Cells During Wound Healing.

J Ocul Pharmacol Ther 2017 04 22;33(3):132-140. Epub 2017 Feb 22.

Ocular Trauma Task Area, U.S. Army Institute of Surgical Research , JBSA Fort Sam Houston, San Antonio, Texas.

Purpose: To characterize the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) by induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. We hypothesize that iPS-RPE secretes mediators of tissue remodeling such as MMPs and TIMPs to promote migration and proliferation of cells during wound healing.

Methods: iPS-RPE was grown on transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Conditioned media were collected from the apical and basolateral sides of the transwells every 72 h for 12 days. The media were analyzed by multiplex ELISA assays to detect secreted MMPs and TIMPs. Activity assays were performed to detect the active form of MMP-2 in conditioned media.

Results: MMP-2 and TIMP-1, -2, -3, and -4 were detected in conditioned media from iPS-RPE. The proteins were found to be secreted in a polarized manner. The apical secretion and activation of MMP-2 was elevated from days 3 to 12 after wounding. TIMP-1, -2, -3, and -4 were detected in conditioned media from both the apical and basolateral sides of wounded cells. Apical secretion of all 4 TIMPs increased within 3 days after wounding.

Conclusions: These results indicate that iPS-RPE secretes MMP-2 and all 4 TIMPs in a polarized manner. After wounding, apical secretion of MMP-2 was higher compared to control. Apical secretion of all 4 TIMPs increased compared to control, while only TIMP-1 showed increased basolateral secretion compared to control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/jop.2016.0070DOI Listing
April 2017

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing.

Invest Ophthalmol Vis Sci 2016 Aug;57(10):4428-4441

Ocular Trauma Task Area US Army Institute of Surgical Research, Joint Base San Antonio-Fort Sam Houston, Texas, United States.

Purpose: The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing.

Methods: iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured.

Results: iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells.

Conclusions: These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1167/iovs.16-19192DOI Listing
August 2016

Recombinant Xeno-Free Vitronectin Supports Self-Renewal and Pluripotency in Protein-Induced Pluripotent Stem Cells.

Tissue Eng Part C Methods 2016 02 15;22(2):85-90. Epub 2016 Jan 15.

1 Ocular Trauma Task Area, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram, maintain, and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently, human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4, c-Myc, KLF4, and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products, we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions, piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm, mesoderm, and endoderm lineages in vitro. In conclusion, recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEC.2015.0180DOI Listing
February 2016

Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium.

Cancer Inform 2014 15;13(Suppl 5):25-35. Epub 2014 Oct 15.

Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA. ; South Texas Center for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX, USA.

The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129-5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA-target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4137/CIN.S14074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4218680PMC
November 2014

MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

J Vis Exp 2014 Jun 24(88):e51589. Epub 2014 Jun 24.

Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston;

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/51589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204998PMC
June 2014

Synergistic killing effect of chloroquine and androgen deprivation in LNCaP cells.

Biochem Biophys Res Commun 2012 Aug 20;425(2):150-6. Epub 2012 Jul 20.

Department of Biochemistry and Molecular Biology and UNM Cancer and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.

Modulation of autophagy is a new paradigm in cancer therapeutics. Recently a novel function of chloroquine (CLQ) in inhibiting degradation of autophagic vesicles has been revealed, which raises the question whether CLQ can be used as an adjuvant in targeting autophagic pro-survival mechanism in prostate cancer (PCa). We previously showed that autophagy played a protective role during hormone ablation therapy, in part, by consuming lipid droplets in PCa cells. In addition, blocking autophagy by genetic and pharmacological means in the presence of androgen deprivation caused cell death in PCa cells. To further investigate the importance of autophagy in PCa survival and dissect the role of CLQ in PCa death, we treated hormone responsive LNCaP cells with CLQ in combination with androgen deprivation. We observed that CLQ synergistically killed LNCaP cells during androgen deprivation in a dose- and time-dependent manner. We further confirmed that CLQ inhibited the maturation of autophagic vesicles and decreased the cytosolic ATP. Moreover, CLQ induced nuclear condensation and DNA fragmentation, a hallmark of apoptosis, in androgen deprived LNCaP cells. Taken together, our finding suggests that CLQ may be an useful adjuvant in hormone ablation therapy to improve the therapeutic efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2012.07.054DOI Listing
August 2012

Autophagy regulates lipolysis and cell survival through lipid droplet degradation in androgen-sensitive prostate cancer cells.

Prostate 2012 Sep 31;72(13):1412-22. Epub 2012 Jan 31.

Department of Biochemistry and Molecular Biology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA.

Background: Androgen deprivation therapy, one of the standard treatments for prostate cancer (PCa) induces apoptosis, as well as autophagy in androgen-responsive PCa cells. As autophagy can promote either cell survival or death, it is important to understand its role in PCa treatment. The objective of this study was to elucidate the function of autophagy in lipid droplet (LD) homeostasis and survival in androgen-sensitive PCa cells.

Methods: To produce androgen deprivation, charcoal filtered serum or the androgen inhibitor casodex were used in LNCaP and LAPC4 cells. Autophagy was monitored by immunofluorescence/confocal microscopy and immunoblot analysis. Levels of intracellular LDs and triacyglycerols after the inhibition of autophagy by 3-methyladenine, bafilomycin A(1) , or si-ATG5 were quantified by three independent methods, Oil Red O staining, triacyglycerols lipase assay, and nuclear magnetic resonance.

Results: Androgen deprivation induced autophagy and the depletion of LDs in both of the androgen-sensitive PCa cell lines examined, whereas the blockage of autophagy by pharmacological or genetic means inhibited LD degradation and therefore lipolysis and cell growth. In addition, under androgen deprivation, increased colocalization of LDs and autophagic vesicles was observed in LNCaP cells, which can be further enhanced by blocking the autophagic flux.

Conclusion: Autophagy mediates LD degradation and lipolysis in androgen-sensitive PCa cells during androgen deprivation which aids the survival of PCa cells during hormone therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.22489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418419PMC
September 2012

Alpha-tocopherol transfer protein deficiency in mice causes multi-organ deregulation of gene networks and behavioral deficits with age.

Ann N Y Acad Sci 2004 Dec;1031:109-26

Center for Comparative Respiratory and Medicine, Department of Internal Medicine, University of California, Davis, CA 95616, USA.

Functions of alpha-tocopherol (alpha-T) in vivo, other than those for fertility in females, are intensely debated. The discovery of alpha-T deficiency in patients with ataxia (AVED) followed by the identification of mutations in the gene encoding alpha-tocopherol transfer protein (TTP) in AVED patients demonstrates an essential role of alpha-T and TTP for normal neurological function. alpha-T molecular targets that account for alpha-T-sensitive neurological dysfunction remain to be discovered. We have used high-density oligonucleotide arrays to search for putative alpha-T-sensitive genes in the CNS and other tissues in an in vivo model of alpha-T deficiency imposed at birth by the deletion of the TTP gene in mice. Repression of genes affecting synaptic function and myelination and induction of genes for neurodegeneration in the motor cortex of alpha-T-deficient mice were identified. The expression of retinoic acid-related orphan receptor alpha (ROR-alpha) was repressed in the cortex and adrenal glands of TTP-deficient mice. Deficiency of ROR-alpha causes ataxia in mice and may account for ataxia in AVED patients. These observations suggest that some of the actions of alpha-T are mediated by the transcription factor ROR-alpha. The behavior of young TTP-null mice was essentially normal, but older mice showed inactivity, ataxia, and memory dysfunction. mRNA profiles of old alpha-T-deficient cerebral cortices are compatible with repressed activity of oligodendrocytes and astrocytes. In conclusion, gene-expression profiling studies have identified novel alpha-T-modulated genes and cells in the CNS that may be causatively linked with delayed neurodegeneration and age-related decline in behavioral repertoires.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1196/annals.1331.012DOI Listing
December 2004