UT, MD Anderson, Science Park - Research Division Side Populations

CD34⁺ Hematopoietic Progenitor Cells

Claudio J. Conti, DVM, Ph.D. and Marcela Bolontrade

Research Core 3: Molecular Genetics & Environmental Carcinogenesis

Mouse Bone Marrow

Hematopoietic Progenitor Cells (HPC) are self renewing, pluripotent stem cells found in pheripheral blood and bone marrow. (The richest source is in umbilical cord.) Their ability to differentiate into other cell types is the basis for engrafting (transplant) whole immune systems in patients with Immunological cancers and other blood disorders. The International Society of Hematotherapy and Graft Engineering (ISHAGE) established the guidelines for single platform CD34⁺ enumeration of HPC which display a low side-scatter profile and dim CD45 expression. This rapid, accurate quantitative analysis usually incorporates 7AAD to discriminate live from dead cells and is amenable to other antigenic markers to analyze CD34⁺ subsets.

References

Sutherland, D.R., Anderson, L., Keeney, M., Nayar, R., and Chin-Lee, I. (1996) The ISHAGE Guidelines for CD34⁺ Cell Determination by Flow Cytometry. J. Hematother. 5:3 p. 213-226.

Side Population (SP) Phenotype

Research Core 5: Mechanisms of Disease Prevention

Jianjun Zhou, Ph.D. and Dean Tang, Ph.D.

Mouse Primary Keratinocytes stained with Hoechst 33342.

Hangwen Li and Dean Tang, Ph.D.

HL-60 cells and HL-60 cells constitutively over expressing the Doxorubicin resistance gene* were stained with Hoechst 33342.

Collene Jeter, Ph.D. and Dean Tang, Ph.D.

MCF-7 Breast Carcinoma Cell Line

The vital fluorescent dye Hoechst 33342 readily permeates live cells but in a small population of cells is differentially pumped out by the p-glycoprotein pump. In hematopoietic stem cells this pump is active and has been associated with adenosine triphosphate binding cassette (ABC) transporter genes Bcrp1 (ABCG2) and/or Mdr1a/1b of which p-glycoproteins are a product. This mechanism is central to the multi drug resistance displayed by tumor cells refractory to chemotherapy.

*Special thanks to Michael Andreeff, M.D., Ph.D. & the M.D. Anderson Flow Cytometry & Imaging Core for their valuable advice and assistance.

References

LaLande, M.E., Ling, V., and Miller, R.G. (1981) Hoechst 33342 dye uptake as a probe of membrane permeability changes in mammalian cells. PNAS 78:1 p. 363-367.

Bunting, K.D., Zhou, S., Lu, T., and Sorrentino, B.P. (2000) Enforced P-glycoprotein pump function in murine bone marrow cells results in expansion of side population stem cells in vitro and repopulating cells in vivo. Blood 96:3, p. 902-909

Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., and Sorrentino, B.P. (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med. 7:9, p. 1028-1034

Zhou, S., Morris, J.J., Barnes, Y., Lan, L. Schuetz, J.D., and Sorrentino, B.P. (2002) Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. PNAS 99:19, p. 12339-12344

Abbott, B.L., Colapietro, A., Barnes, Y., Marini, F., Andreeff, M., and Sorrentino, B.P. (2002) Low levels of ABCG2 expression in adult AML blast samples. Blood 100:13, p. 4594-4601

Protocol

M.D. Anderson Flow Cytometry & Imaging Core

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Last updated: 20-November-06