Defining Molecular Phenotypes of Mesenchymal and hematopoietic Stem Cells derived from Peripheral blood of Acute Lymphocytic Leukemia patients for regenerative stem cell therapy

Abstract

Acute Lymphocytic Leukemia (ALL) is a clonal myeloid disorder affecting all age groups, characterized by accumulation of immature blast cells in bone marrow and in peripheral blood. Autologous Bone Marrow Transplantation is a present treatment for cure of ALL patients, which is very expensive, invasive process and may have possibility of transplantation of malignant stem cells to patients. In the present study, we hypothesized to isolate large number of normal Mesenchymal & Hematopoietic stem cells from peripheral blood of ALL patients, which will be further characterized for their normal phenotypes by using specific molecular stem cell markers. This is the first study, which defines the existing phenotypes of isolated MSCs and HSCs from peripheral blood of ALL patients. We have established three cell lines in which two were Mesenchymal stem cells designated as MSCALL and MSCnsALL and one was suspension cell line designated as HSCALL. The HSCALL cell line was developed from the lymphocyte like cells secreted by MSCALL cells. Our study also showed that MSCALL from peripheral blood of ALL patient secreted hematopoietic stem cells in vitro culture. We have characterized all three-cell lines by 14 specific stem cell molecular markers. It was found that both MSC cell lines expressed CD105, CD13, and CD73 with mixed expression of CD34 and CD45 at early passage whereas, HSCALL cell line expressed prominent feature of hematopoietic stem cells such as CD34 and CD45 with mild expression of CD105 and CD13. All three-cell lines expressed LIF, OCT4, NANOG, SOX2, IL6, and DAPK. These cells mildly expressed COX2 and did not express BCR-ABL. Overall it was shown that isolated MSCs and HSCs can be use as a model system to study the mechanism of leukemia at stem cell level and their use in stem cell regeneration therapy for Acute Lymphocytic Leukemia.

Keywords: Acute Lymphocytic Leukemia; HSCs; MSCs; Peripheral blood cells; Stem cell markers.

Figure 1

shows confluent MSC_ALL cells having spindle shaped morphology observed 20 days after first adherent cells were seen

Figure 2

shows Cell growth rate study graph of MSC_ALL (left) and HSC_ALL (right) cell lines

Figure 3

shows cells that are being produced (HSC_ALL) from adherent cells and could be seen coming into the suspension

Figure 4

shows clones that were formed by HSC_ALL cells in suspension culture

Figure 5

shows Geimsa Stained HSC_ALL cells, which shows mononuclear cells with large nucleus and scanty

Figure 6

shows Large flattened MSCns_ALL cells, which stops secreting HSCs observed after 6 weeks in culture

Figure 7

shows expression of molecular markers CD105, CD13, CD73, CD34 and CD45 in MSC_ALL, MSCns_ALL & HSC_ALL cell lines.

Figure 8

shows expression of molecular markers OCT4, NANOG, SOX2, LIF and KERATIN18 in MSC_ALL, MSCns_ALL & HSC_ALL cell lines.

Figure 9

shows expression of molecular markers DAPK, COX2, BCR-ABL and IL6 in MSC_ALL, MSCns_ALL & HSC_ALL cell lines.