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Differentiation of marrow stromal cells into photoreceptors in the rat eye.

ABSTRACT: Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific markers rhodopsin, opsin, and recoverin in vitro. CD90+ cells were either transduced with recombinant adeno-associated virus expressing green fluorescent protein (GFP) or bromodeoxyuridine (BrdU) labeled and then injected into the subretinal space of adult Royal College of Surgeons rats. Fundus photography and angiography showed no adverse effects of CD90+ MSC transplantation. GFP-expressing cells or BrdU-positive cells covered approximately 30% of the entire retinal area. By 2 weeks after injection, CD90+ MSCs integrated into the host retina, forming structures similar to the photoreceptor layer and expressed a photoreceptor-specific marker. No teratoma formation was observed in the recipient retina. The subretinally delivered CD90+ MSCs did not stain for proliferating cell nuclear antigen, indicating that they primarily undergo differentiation rather than proliferation. In addition, we established that transplanted cells can attract synaptic vesicles and hence are potentially capable of signal transduction. This study demonstrates for the first time the partial differentiation of adult CD90+ MSCs into photoreceptors in vitro and in vivo. Our results establish a proof of concept for CD90+ MSC differentiation with autologous transplantation, which may provide a promising therapeutic strategy for the treatment of some forms of genetically inherited retinal degenerations.

SUBMITTER: Kicic A

PROVIDER: S-EPMC6740611 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Differentiation of marrow stromal cells into photoreceptors in the rat eye.

Kicic Anthony A Shen Wei-Yong WY Wilson Ann S AS Constable Ian J IJ Robertson Terry T Rakoczy P Elizabeth PE

The Journal of neuroscience : the official journal of the Society for Neuroscience 20030801 21

Retinal degenerations and dystrophies are the major causes of genetically inherited blindness that are characterized by the apoptotic death of the photoreceptor cell layer of the retina. To date, no treatment exists for these diseases and only recently have they been considered as candidates for gene and stem cell therapies. Here we report the ability of adult CD90+ marrow stromal cells (MSCs) to be induced by activin A, taurine, and EGF into cells (20-32%) expressing photoreceptor-specific mark ...[more]

PMID: 12944502

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Project description:FK506 (also known as tacrolimus) is a potent immunosuppressive agent that is widely used in the treatment of graft-rejection and autoimmune diseases. FK506 has attracted additional attention owing to its potential role in osteogenic differentiation and bone formation. MicroRNAs (miRNAs) have been demonstrated to serve important roles in the regulation of osteogenic differentiation; however, identification of specific miRNAs and their roles in regulating FK506‑induced osteogenic differentiation have been poorly examined. In the present study, osteodifferentiation of rat bone marrow stromal cells (BMSCs) was induced with varying concentrations of FK506 (5‑5,000 nM) for 3, 7 and 14 days. Differentially expressed miRNAs were profiled using miRNA array, verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and subjected to gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results from the present study identified a subset of miRNAs that were differentially expressed, of which five upregulated miRNAs (miR‑106b‑5p, miR‑101b‑3p, miR‑193a‑3p, miR‑485‑3p and miR‑142‑3p) and four downregulated miRNAs (miR‑27a‑3p, miR‑207, miR‑218a‑2‑3p and let‑7a‑5p) were confirmed by RT‑qPCR. GO and KEGG analysis revealed that the predicted target genes of these miRNAs are involved in multiple biological processes and signaling pathways, including cell differentiation and the mitogen‑activated protein kinase (MAPK) signaling pathway. Verification of the miRNA‑target genes revealed that Smad5, Jagged 1 and MAPK9 were significantly upregulated, whereas Smad7, BMP and activin membrane‑bound inhibitor, and dual‑specificity phosphatase 2 were significantly downregulated during FK506‑induced osteodifferentiation. The present study may provide an experimental basis for further research on miRNA functions during FK506‑induced osteogenic differentiation in rat BMSCs.

Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Project description:Purpose:This study is aimed at investigating the phenotype, differentiation potential, immunomodulatory properties, and responsiveness of saphenous vein vessel wall-derived mesenchymal stromal cells (SV-MSCs) to various TLR ligands and proinflammatory cytokines, as well as comparing their features to those of their bone marrow-derived counterparts (BM-MSCs). Methods:SV-MSCs were isolated by enzymatic digestion of the saphenous vein vessel wall. Phenotype analysis was carried out by flow cytometry and microscopy, whereas adipogenic, chondrogenic, and osteogenic differentiation potentials were tested in in vitro assays. For comparative analysis, the expression of different stemness, proliferation, and differentiation-related genes was determined by Affymetrix gene array. To compare the immunomodulatory properties of SV-MSCs and BM-MSCs, mixed lymphocyte reaction was applied. To investigate their responses to various activating stimuli, MSCs were treated with TLR ligands (LPS, PolyI:C) or proinflammatory cytokines (TNF?, IL-1?, IFN?), and the expression of various early innate immune response-related genes was assessed by qPCR, while secretion of selected cytokines and chemokines was measured by ELISA. Results:The isolated SV-MSCs were able to differentiate into bone, fat, and cartilage cells/direction in vitro. SV-MSCs expressed the most important MSC markers (CD29, CD44, CD73, CD90, and CD105) and shared almost identical phenotypic characteristics with BM-MSCs. Their gene expression pattern and activation pathways were close to those of BM-MSCs. SV-MSCs showed better immunosuppressive activity inhibiting phytohemagglutinin-induced T lymphocyte proliferation in vitro than BM-MSCs. Cellular responses to treatments mimicking inflammatory conditions were comparable in the bone marrow- and saphenous vein-derived MSCs. Namely, similar to BM-MSCs, SV-MSCs secreted increased amount of IL-6 and IL-8 after 12- or 24-hour treatment with LPS, PolyI:C, TNF?, or IL-1?, compared to untreated controls. Interestingly, a different CXCL-10/IP-10 secretion pattern could be observed under inflammatory conditions in the two types of MSCs. Conclusion:Based on our results, cells isolated from saphenous vein vessel wall fulfilled the ISCT's (International Society for Cellular Therapy) criteria for multipotent mesenchymal stromal cells, and no significant differences in the phenotype, gene expression pattern, and responsiveness to inflammatory stimuli could be observed between BM-MSCs and SV-MSCs, while the latter cells have more potent immunosuppressive activity in vitro. Further functional assays have to be performed to reveal whether SV-MSCs could be useful for certain regenerative therapeutic applications or tissue engineering purposes.

Dataset Information

Differentiation of marrow stromal cells into photoreceptors in the rat eye.

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Differentiation of marrow stromal cells into photoreceptors in the rat eye.

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