title: Gel based in vitro 3D model exploring the osteocytic potentiality of human CD34+stem cells.
abstract: 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.
authors: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India.
authors: Lokanathan
authors: L
authors: Srikanth
authors: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India.
authors: Manne Mudhu
authors: MM
authors: Sunitha
authors: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India.
authors: Pasupuleti Santhosh
authors: PS
authors: Kumar
authors: Department of Hematology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.
authors: Chodimella
authors: C
authors: Chandrasekhar
authors: Department of Neurology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India.
authors: Bhuma
authors: B
authors: Vengamma
authors: Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India. sarmasvims@gmail.com.
authors: Potukuchi Venkata Gurunadha Krishna
authors: PV
authors: Sarma
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D005819:Genetic Markers / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008954:Models, Biological; D010011:Osteocytes / Q000166:cytology*; D010012:Osteogenesis; D013234:Stem Cells / Q000166:cytology* / Q000276:immunology
chemical_list: D018952:Antigens, CD34; D005819:Genetic Markers; D007377:Interleukin-3; D016179:Granulocyte Colony-Stimulating Factor
keywords: Human CD34+ stem cells; Osteoblasts; RANKL; Runx2; Sclerostin