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Examines in situ apoptosis of bone marrow mononuclear cells (BMMNC) in the BM biopsies of aplastic anemia patients. Methodology used; Results of the study on BMMNC.

We have evaluated the cardiomyogenic potential of clonal populations of human bone marrow mesenchymal stem cells (BM-MSC). Four rapidly proliferating clones of BM-MSC were obtained from the BM of a healthy donor which were then treated with 5-azacytidine and evaluated for the expression of GATA-4, NKx-2.5, FOG-2, TDGF-1, β-MHC, MEF2D and NPPA genes and cTnT, Desmin and β-MHC proteins. Of the four clones (i) Clone-1 had high expression of GATA-4 (1.89 fold (p<0.05), Nkx2.5 (2.29 fold; p<0.05), FOG2 (2.76 fold; p<0.05), TDGF1 (6.97 fold, p<0.005), βMHC (10.22 fold; p<0.005), MEF-2D (1.91 fold; p<0.005) and NPPA (1.65 fold; p<0.005); (ii) clone-2 had up-regulation of Nkx2.5 (1.98 fold; p<0.05) but down-regulation of rest of the genes; (iii) clone-3 had up-regulation of Nkx2.5 (2.11 fold; p<0.05), TDGF1 (1.88 fold; p<0.05), MEF-2D (1.30 fold; p<0.05) and NPPA (1.21 fold; p<0.05), down regulation of GATA-4 and Fog-2 but no change in βMHC gene; and (iv) clone-4 had up-regulation of MEF-2D (1.17 fold; p<0.05) and down regulation of GATA-4, Nkx2.5 but no change in other genes compared to untreated cells of the clones. At the protein level, clone-1 expressed cTnT, Desmin, and βMHC; clone-2 Desmin only while clones-3 and 4 each expressed cTnT, Desmin, and βMHC. Our data shows that BM-MSC are a heterogenous population of stem cells with sub-populations exhibiting a marked difference in the expression of cardiac markers both at gene and protein levels. This highlights that administering selected sub-populations of BM-MSC with a cardiomyogenic potential may be more efficacious than whole population of cells for cardiac regeneration.

Aim: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis., Methods: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3(rd) passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot., Results: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05)., Conclusion: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.

Aim: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC)., Methods: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 and human leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR., Results: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82% vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34), GFAP (1.12), Tau (1.08), MAP-1B (0.92), MAP-2 (1.14) and NSE (0.4) (P < 0.001 for all)., Conclusion: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

Aim: To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells (fC-MSC) and their differentiation into cells of all the germ layers., Methods: Ten independent cultures of rat fC-MSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3, 6, 15 and 21. The phenotypic markers CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR were analyzed by flow cytometry. The expression of embryonal markers Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA 1-81 were studied by immunocytochemistry. The fC-MSC treated with specific induction medium were evaluated for their differentiation into (1) adipocytes and osteocytes (mesodermal cells) by Oil Red O and Alizarin Red staining, respectively, as well as by expression of lipoprotein lipase, PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction (RT-PCR); (2) neuronal (ectodermal) cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry; and (3) hepatocytic (endodermal) cells by expression of albumin by RT-PCR and immunocytochemistry, glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant., Results: The fC-MSC expressed CD29, CD73, CD90, CD105, CD166 but lacked expression of CD31, CD34, CD45 and HLA-DR. They expressed embryonal markers, viz. Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-81 but not TRA-1-60. On treatment with specific induction media, they differentiated into adipocytes and osteocytes, neuronal cells and hepatocytic cells., Conclusion: Our results together suggest that fC-MSC are primitive stem cell types with a high degree of plasticity and, in addition to their suitability for cardiovascular regenerative therapy, they may have a wide spectrum of therapeutic applications in regenerative medicine.

We have investigated intracellular production by T cells and plasma levels of TNF-alpha, IL-2 and IFN-gamma in 12 active and 10 inactive Takayasu's arteritis (TA) patients and 12 healthy controls. The active TA compared to inactive TA and controls had higher TNF-alpha (52.7 +/- 22.3% vs. 32.9 +/- 14.2% and 35.2 +/- 14.5%, respectively; P = 0. 020), lower IL-2 (19.6 +/- 13.2% vs. 36.1 +/- 10.1% and 31.2 +/- 10.3%, respectively; P = 0.010) and comparable IFN-gamma (38.6 +/- 13.9% vs. 34.2 +/- 12.4% and 34.9 +/- 11.1%, respectively; P = 0.581) producing CD3+ T cells. There was no difference in the plasma levels of the cytokines between active TA, inactive TA and controls (TNF-alpha: 79.1 +/- 94.5 vs. 72.9 +/- 120.0 and 9.5 +/- 6.7 pg/ml, P = 0.110; IL-2: 4.3 +/- 4.8 vs. 6.6 +/- 4.7 and 8.6 +/- 4.5 pg/ml, P = 0.094 and IFN-gamma: 10.1 +/- 11.3 vs. 8.8 +/- 8.7 and 8.2 +/- 6.5 pg/ml, P = 0.871, respectively). The data show an important role of these high TNF-alpha and low IL-2 producing T cells in TA.

Interleukin-8 (IL-8), a CXC chemokine, is also a potent inhibitor of myelopoiesis, the hematopoietic process that is severely impaired in aplastic anemia (AA). To elucidate its role in the disease, we have investigated levels of IL-8 by quantitative enzyme-linked immunosorbent assay in bone marrow and peripheral blood plasma of 27 AA patients and in the marrow of 16 controls and blood of 20 controls. Significantly increased levels of IL-8 were observed in the marrow and blood of patients as compared to controls (470.4 +/- 549.6 vs. 37.5 +/- 30.3; P < 0.0001) and (247.3 +/- 286.3 vs. 7.9 +/- 5.5; P < 0.0001), respectively. Among the patients, the IL-8 levels were higher in patients with severe AA than those with nonsevere AA in the marrow (568.8 +/- 586.9 vs. 126.3 +/- 102.5; P < 0.005) as well as in the blood (296.6 +/- 305.5 vs. 75.0 +/- 84.4; P < 0.008) plasma. The marrow and blood of 74% (20/27) of the patients had increased levels of IL-8 compared to 12% (2/16; P < 0.001) and 10% (2/20, P < 0.001) of the controls, respectively. These results suggest that IL-8 may have an important role in the pathogenesis of AA.

Objective: To investigate the prevalence of antimonocyte antibodies (AMA) in Takayasu's arteritis (TA) and their relationship with disease activity., Methods: IgG-AMA were studied in the sera of 60 patients with TA (29 active disease, 31 inactive) and 43 controls by a cellular ELISA using glutaraldehyde fixed U-937 cells or peripheral blood monocytes as antigen. Relationship of AMA with disease activity was evaluated by measuring titers of these antibodies in followup sera of 15 AMA positive patients with active TA undergoing immunosuppressive therapy., Results: Twenty-six of 60 TA patients (43%) compared to 4 of 43 controls (9%) (p < 0.001) and 20 of 29 patients with active disease (69%) compared to 6 of 31 patients with inactive disease (19%) (p < 0.001) were positive for AMA. The antibody titers were significantly higher in patients with active disease than those with inactive disease (0.396 +/- 0.172 vs 0.232 +/- 0.096; p < 0.001). In the followup study of 15 patients with active disease who received immunosuppressive therapy, we observed normalization of AMA titers in 6 of the 7 patients who became inactive, compared to only one of the 8 patients whose disease remained active during followup (p < 0.01)., Conclusion: AMA are present in a significant proportion of patients with TA and correlate with disease activity, suggesting a possible pathogenic role of these antibodies in TA.