Down-regulation of PAX2 promotes in vitro differentiation of podocytes from human CD34+ cells.

Podocytes are major kidney cells that help in glomerular filtration and any damage or loss is a major event in the progression of kidney diseases. Understanding podocytes development will help in designing therapeutic strategies against these renal diseases. Therefore, in vitro generation of podocytes from adult hematopoietic CD34+ stem cells is explored in the present study. Apheretically, isolated human HSCs from peripheral blood showed the presence of CD34 surface glycoprotein through immunocytochemistry (ICC) and flowcytometry. Initially, these HSCs were induced with activin-A (10 ng/ml), retinoic acid (RA) (10 ng/ml) and bone morphogenic protein (BMP-7) (2.5 ng/ml) for 5 days. Transdifferentiation of HSCs to podocytes through intermediate mesoderm was studied with positive selection of Osr1+ cells. Subsequently, thus-obtained Osr1+ cells were induced further with activin-A (10 ng/ml), RA (10 ng/ml), BMP-7 (2.5 ng/ml), EGF (30 ng/ml) and bFGF (30 ng/ml) for 9 days. Distinct cobblestone morphological changes were observed on staining with Leishman's stain. Consequently, differentiated cells were immunopositive for anti-podocin, anti-synaptopodin and anti-GLEPP1 monoclonal antibodies. These cells showed expression of early podocyte markers PAX2 and Wt1 at day 3 followed by day 6 and mature podocyte markers NPHS1, SULT1B1, NPHS2 and Synaptopodin at day 9. Interestingly, on day 9, diminished expression of PAX2 was noted. Differentiated cells showed high tyrosine kinase activity signifying that phosphorylation controls slit diaphragm proteins. Synaptopodin regulates the integrity of cytoskeleton and cell motility of podocytes and this phenomenon was confirmed through scratch assay using agarose molds that showed high cell mobility and migration. These findings establish HSCs as ideal candidates for regenerative therapies of damaged podocytes.

Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

BACKGROUND: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. METHODS: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. RESULTS: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. CONCLUSION: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Gel based in vitro 3D model exploring the osteocytic potentiality of human CD34+ stem cells.

Osteocytic potentiality of human CD34+ stem cells explored in the present study by generating in vitro agarose gel 3D model to understand the bone ossification process. The G-CSF and IL-3 mobilized human CD34+ stem cells isolated apheretically from donor peripheral blood and purity of the cells was assessed by FACS and immunocytochemical (ICC) studies. The CD34+ stem cells were cultured in gel based 3D model with osteogenic stimulating medium for 21 days. The transition stages from undifferentiated to differentiated osteocytes through osteoblasts were studied with expression markers Differentiated cells at Day 7 showed positive reactivity with monoclonal anti-Runx2, an early osteoblastic marker. qPCR expression analysis showed early and mature osteoblastic markers like RUNX2, Osterix, RANKL, along with osteocyte markers SPARC, Sclerostin. While poor expression of OSCAR genes was observed apart from conspicuous expression of alkaline phosphatase. The expression of sclerostin and SPARC suggests that these differentiated cells are behaving like true osteocytes, sclerostin expression causes transformation of osteoblast into osteocytes and negligible expression of OSCAR, RANK, NFATc and cathepsin K genes explains there are no osteoclasts in the differentiated culture. These cells showed positive reaction with Alizarin red stain indicating expression of calcium bound bone morphogenic proteins like osteonectin. All these results clearly confirm the human CD34+ stem cells possess unique osteogenic differentiation potential and can be used in the early regeneration of injured bone.

In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic ß-cells.

Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into ß-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific ß-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained ß-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into ß-cells of islets of langerhans.

In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells.

OBJECTIVES: Human CD34(+) stem cells differentiated into type-II pneumocytes in Dulbecco's modified Eagle medium (DMEM) having hydrocortisone, insulin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and bovine serum albumin (BSA), expressing surfactant proteins-B (SP-B) and C (SP-C), alkaline phosphatase (ALP) and lysozyme. RESULTS: FACS-enumerated pure CD34(+) cells, isolated from human peripheral blood, were cultured in DMEM and showed positive reaction with anti-human CD34 monoclonal antibodies in immunocytochemistry. These cells were cultured in DMEM having hydrocortisone, insulin, FGF, EGF and BSA (HIFEB-D) medium having an air-liquid interface. They differentiated into type-II pneumocytes with expression of SP-B and SP-C genes and disappearance of CD34 expression as assessed using real-time PCR. In reverse transcription-PCR amplicons showed 208 and 907 bp confirming SP-B and SP-C expressions. These cells expressed ALP with an activity of 1.05 ± 0.09 mM ml(-1) min(-1) and lysozyme that killed E. coli. CONCLUSION: The successful differentiation of human CD34(+) stem cells into type-II pneumocytes, and transplantation of such cells obtained from the patient's stem cell could be the futuristic approach to regenerate diseased lung alveoli.

Role of STPK in Maintaining the Quiescent Nature of Human CD34+ Stem Cells.

Haematopoietic stem cell normally exists in the hypoxic niche of bone marrow and in this high anaerobic condition phosphorylation is vital in understanding the stemness of these stem cells in bone marrow. Analysis of human aldehyde dehydrogenase (ALDH) and isocitrate dehydrogenase (IDH) we have observed the presence of serine threonine protein kinase (STPK) sites in the protein sequence of these enzymes conferring that functioning of ALDH and IDH is regulated largely by STPK through phosphorylation. Human CD34+ stem cells and mononuclear cells as a control isolated from peripheral blood and were propagated in DMEM media at 5% CO2, 95% humidity and at 37°C. Thus obtained cells showed high enzyme activity for STPK, ALDH and low enzyme activity for IDH in CD34+ cells compare to control cells. These results were concurred with qRT-PCR studies with high gene expression levels of hypoxia inducing factor-1-alpha (HIF1α), STPK, ALDH and low IDH expression in CD34+ cells while normalized with ß-actin. In addition the phosphorylating sites on ALDH and IDH proteins were identified and their importance in maintaining the anaerobic conditions in HSCs was demonstrated. In view of the importance of STPK signalling in the present study mechanism in cell division was addressed with phosphorylation of key regulating enzymes in the metabolic pathway of cell cycle was explored.

Anaerobic Glycolysis and HIF1α Expression in Haematopoietic Stem Cells Explains Its Quiescence Nature.

Metabolic alteration that a stem cell undergoes during proliferation and quiescence are decisive. These cells survive in extreme hypoxic environment that prevails in bone marrow. The present study is aimed to understand this nature in hematopoietic stem cells. These stem cells were mobilized from bone marrow into peripheral blood by giving G-CSF at a concentration of 5 µg/Kg/d and the cells were isolated by apheresis technique. The morphological analysis of these cells using Giemsa stain and SEM showed presence of only single type of cells with conspicuous nuclei, the hematopoietic nature was assessed by the presence of CD34, a glycoprotein using anti-CD34 monoclonal antibodies. The ICC results revealed presence of CD34 marker further; pure population of CD34+ stem cells was described by FACS. These cells were cultured separately in DMEM having 5.5mM, 11.1mM and 25mM glucose respectively. In these cells GK, PK and L-LDH enzyme activities were estimated which showed increased activities at 5.5mM glucose concentration and further elevation of glucose concentration the activities were fallen considerably. Similarly, qPCR analysis of HIF1α and GAPDH genes showed very high expression of HIF1α at 5.5mM glucose concentration which reduced with increased glucose concentration. While GAPDH gene expression enhanced on elevation of glucose concentration. Thus, these results indicate high HIF1α expression in low glucose condition with improved anaerobic glycolysis seems to be one of the key factors in maintaining the quiescent state of CD34+ stem cells.

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness.

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H(+) secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding ß1 subunit of H(+)-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H(+) ions contributing to dRTA with sensorineural deafness.