Long-Term Coculture of Adult Human Healthy Donor CD34+ Cells Shows Elevation of Telomeres in a Subgroup of Donors Independent of Age, Stem Cell Function and Telomerase Activity.

Abstract

Long-term cytokine-supplemented or stromal cocultures of human CD34+ cells, particularly from cord blood (CB), show expansion of hematopoietic progenitors and stem cells. Ultimately, however cultures decline and terminally differentiate. Despite upregulation of telomerase activity in proliferating primitive hematopoietic cells, telomere shortening has generally been reported in long-term cultures of CB, bone marrow or G-CSF mobilized peripheral blood CD34+ cells. In earlier reports, we described a long-term culture of hematopoietic stem cells on a murine OP9 bone marrow stroma cell line transfected with an adenovector expressing thrombopoietin, which allowed an extensive proliferation and self-renewal of CB CD34+ cells for 4–5 months with sustained elevation of telomerase activity and without concomitant significant telomere shortening (Blood, 2004). Here, we evaluated adult healthy donor peripheral blood (PB) CD34+ cells in the same OP9/Tpo coculture system. To determine progenitor and stem cell production, standard CFC and 2ndry cobblestone area-forming cell assays (CAFC assayed at 5 weeks on MS5 stroma) were undertaken weekly with suspension cells. In addition telomere length was measured by telomere restriction fragment (TRF) assay, and telomerase activity by TRAP assay on input CD34+ cells, and weekly on culture suspension cells. Maximum total cell, CFC and CAFC production was seen in the first 4 weeks with up to 80-fold expansion in cell count, up to 4-fold expansion in CFC and up to 13-fold expansion in CAFC. Thereafter a continuous decrease in production of cells, CFC 2ndry CAFC was observed and cultures terminated at week 8. Mean telomere length of input PB CD34+ cells was 9,5 ± 0,5 kbp. After 4 weeks in culture, telomere length remained stable (9,6 ± 0,5 kbp). In 3/6 cultures terminated, cultures showed only a slightly decrease of telomere length compared to the input population (0,55 ± 0,1 kbp loss). However, in 2/6 cultures we could demonstrate an elevation of telomeres (+ 0,3 kbp) independent of a rapid loss of telomerase activity in all cultures during the culture period. Furthermore, the elevation of telomeres did not correlate with an enhanced stem/progenitor cell activity. These data confirm earlier results of granulocyte telomere change in myeloma patients following chemotherapy and tandem transplantation, where 154/193 patients showed an expected loss of telomeres during the treatment period, whereas 39/193 patients had an unexpected elevation of telomeres. We could now show in healthy donors that this phenomenon is independent of bone marrow stress due to chemotherapeutic treatment. In conclusion, we could show that the stromal coculture system with OP9/Tpo is highly effective in stem/progenitor cell expansion not only in CB but also in PB CD34+ cells. It is the first culture system, which allows expansion of hematpoietic cells without significant telomere erosion. We furthermore describe for the first time an age-independent healthy donor population which shows telomerase-independent, significant telomere elevation. Further studies have to demonstrate, if this phenomenon is potentially linked to a higher susceptibility for cancer disease.