Differential expression of 2 integrin separates long-term and short-term reconstituting Lin^-/loThy1.1^loc-kit⁺Sca-1⁺ hematopoietic stem cells

¹ Stanford University School of Medicine, Stanford, California

^* To whom correspondence should be addressed. E-mail: amy.wagers{at}joslin.harvard.edu.

	Abstract

Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin^- Thy1.1^loc-kit⁺Sca-1⁺ subset of normal mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically, and perhaps functionally. We previously identified 2 integrin (CD49b) as a surface marker with heterogeneous expression on Lin^-/loThy1.1^loc-kit⁺Sca-1⁺ stem cells. To determine whether differences in 2 expression were indicative of differences in stem cell function, we purified 2^- and 2^hi stem cells by fluorescence activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both 2^- and 2^hi cells could give rise to mature lymphoid and myeloid cells following transplantation into lethally irradiated, congenic recipients. However, 2^hi cells supported hematopoiesis only for a short time (<4 weeks), while 2^- cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that 2^- cells represent a more primitive population than 2^hi cells. Consistent with this idea, 2^-Lin^-/loThy1.1^loc-kit⁺Sca-1⁺ cells exhibited an 6-fold decreased frequency of spleen colony forming units (day 12) versus 2^hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 2^-Lin^-/loThy1.1^loc-kit⁺Sca-1⁺ cells included both 2^- and 2^hi stem cells of donor origin, indicating that 2^hi cells are likely lineal descendents of 2^- cells. Interestingly, 2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of 2 selectively marks a subset of primitive hematopoietic cells that retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.