Probing the mitotic history and developmental stage of hematopoietic cells using single telomere length analysis (STELA).

In most human somatic cells, telomeres shorten as a function of DNA replication. Telomere length is therefore an indirect measure of the replicative history of cells. We measured the telomere lengths at XpYp chromosomes in purified human hematopoietic populations enriched for stem cells (Lin(-)CD34(+)CD38(-)Rho(-)) and successively more mature cells. The average telomere length showed expected length changes, pointing to the utility of this method for classifying novel differentiation markers. Interestingly, the frequency of abruptly shortened telomeres increased in terminally differentiated adult populations, suggesting that damage to telomeric DNA occurs or is not repaired upon hematopoietic differentiation. When Lin(-)CD34(+)CD38(-)Rho(-) cord blood cells were transplanted into immunodeficient mice, the telomeres of the most primitive regenerated human hematopoietic cells lost approximately 3 kb, indicative of more than 30 cell divisions. Further losses in differentiating cells were similar to those observed in pretransplantation cell populations. These results indicate extensive self-renewal divisions of human hematopoietic stem cells are the primary cause of telomere erosion upon transplantation rather than added cell divisions in downstream progenitors.

Improved purification of hematopoietic stem cells based on their elevated aldehyde dehydrogenase activity.

BACKGROUND AND OBJECTIVES: Primitive human hematopoietic cells contain higher levels of aldehyde dehydrogenase (ALDH) activity than their terminally differentiating progeny but the particular stages when ALDH levels change have not been well defined. The objective of this study was to compare ALDH levels among the earliest stages of hematopoietic cell differentiation and to determine whether these could be exploited to obtain improved purity of human cord blood cells with long-term lympho-myeloid repopulating activity in vivo. DESIGN AND METHODS: ALDEFLUOR-stained human cord blood cells displaying different levels of ALDH activity were first analyzed for co-expression of various surface markers. Subsets of these cells were then isolated by multi-parameter flow cytometry and assessed for short-and long-term repopulating activity in sublethally irradiated immunodeficient mice. RESULTS: Most short-term myeloid repopulating cells (STRC-M) and all long-term lympho-myeloid repopulating cells (LTRC-ML) stained selectively as ALDH+. Limiting dilution analysis of the frequencies of both STRC-M and LTRC-ML showed that they were similarly and most highly enriched in the 10% top ALDH+ cells. Removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, or glycophorin A from the ALDH+ low-density fraction of human cord blood cells with low light side-scattering properties yielded a population containing LTRC-ML at a frequency of 1/360. INTERPRETATION AND CONCLUSION: Elevated ALDH activity is a broadly inclusive property of primitive human cord blood cells that, in combination with other markers, allows easy isolation of the stem cell fraction at unprecedented purities.