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

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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.

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Abbreviations

ALP:

Alkaline phosphatase

CD34+ :

Cluster of Differentiation 34

OSCAR:

Osteoclast-associated immunoglobulin-like receptor

OPG:

Osteoprotegerin

RUNX2:

Runt-related transcription factor 2

RANKL:

Receptor activator of nuclear factor kappa-B ligand

SPARC:

Secreted Protein Acidic and Rich in Cysteine

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Acknowledgments

We sincerely acknowledge Sri Venkateswara Institute of Medical Sciences (SVIMS University), India for providing funds to carry out the work. This paper forms a part of Ph. D. thesis work going to be submitted to SVIMS University, Tirupati, Andhra Pradesh, India.

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Authors and Affiliations

  1. Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India

    Lokanathan Srikanth, Manne Mudhu Sunitha, Pasupuleti Santhosh Kumar & Potukuchi Venkata Gurunadha Krishna Sarma

  2. Department of Hematology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

    Chodimella Chandrasekhar

  3. Department of Neurology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

    Bhuma Vengamma

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  1. Lokanathan Srikanth
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  2. Manne Mudhu Sunitha
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  3. Pasupuleti Santhosh Kumar
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  4. Chodimella Chandrasekhar
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  5. Bhuma Vengamma
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  6. Potukuchi Venkata Gurunadha Krishna Sarma
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Correspondence to Potukuchi Venkata Gurunadha Krishna Sarma.

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Srikanth, L., Sunitha, M.M., Kumar, P.S. et al. Gel based in vitro 3D model exploring the osteocytic potentiality of human CD34+ stem cells. Mol Biol Rep 43, 1233–1242 (2016). https://doi.org/10.1007/s11033-016-4053-4

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  • DOI: https://doi.org/10.1007/s11033-016-4053-4

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