An Evidence That Mesenchymal Stem Cells Are Not Replaced by Peripheral Blood Stem Cell Allografts in CML Patients.

Four CML patients were investigated for post transplant chimerism for total marrow (BM) cells and Mesenchymal Stem Cell (MSC) populations purified and propagated from BM cells. Patients were conditioned with BuFluATG and after allogeneic PBPCT were disease free with proven hematological, cytogenetical and genetical remission. For the study 15–30 ml fresh BM aspirates were phenotypically characterized for the presence of cell populations which may contain MSC. We found (median values): 11.7% CD45-CD34− 1.7% CD45-CD34-CD105+, 0.13% CD45-CD34-CD90+ and 0.03% CD45-CD34-CD73+ cells. These BM populations contained 0.75% CD34+ cells. Genetical work showed that BM cells were BCR-ABL negative and their STR informative allele patterns were consistent with those of donors. BM cells were processed as follows:

• incubation with Glycophorin A, CD3, CD14, CD19, CD66b, CD38 antibody cocktail which by cross-linking unwanted cells with red blood cells led to rosette formation (RosetteSep MSC Enrichment Cocktail, StemCell Technology),

• unrosetted cells (MSC enriched) were recovered from the interface after buoyant gradient centrifugation and contained (median values): 68.7% CD45-CD34-, 21.6% CD45-CD34-CD105+, 1.1% CD45-CD34-CD90+ and 0.4% CD45-CD34-CD73+.

MSC enriched BM populations were cultured in Medium for Human MSC with Stimulatory Supplements (StemCell Technology). After 10–14 days CFU-F colonies were scored (median value was 77 CFU-F/10⁶ cells) and cells were further cultured until >90% confluence of fibroblast-like cells were reached (usually 3 to 4 weeks after culture initiation). The cells were detached with 0.05% trypsin-EDTA and studied for STR allele patterns, the presence of BCR-ABL transcripts (at that time cells showed STR alleles of the recipient pattern for the first time - mixed chimerism) and the bulk of cells were further passaged. Usually after 3–4 passages (within 7–8 weeks) when fibroblast-like stromal cell populations reached the level of 3x10⁶ cells, cultures were terminated and the cells were studied. These cells were in (median values) 27.0% CD45-CD34-, 23.8% CD45-CD34-CD105+, 26.5% CD45-CD34-CD90+ and 24.3% CD45-CD34-CD73+. The cells had a fibroblast-like morphology but only 26% had phenotype features of MSC on average. Therefore, the population consisted of MSC and more differentiated cells originated from CFU-F (MSC). RNA and DNA were isolated from the cells propagated for 7–8 weeks from the MSC enriched BM populations were BCR-ABL negative. However, their STR informative allele patterns were consistent with those of the recipients in variance to primary BM cell populations which was in all 4 cases of donor origin.

Conclusions:

• with the use of the RosetteSep MSC enrichment purification system BM cells can be enriched in CFU-F which paralleled with an increase in the proportion of CD45-CD34-, CD45-CD34-CD90+, CD45-CD34-CD73+ and CD45-CD34-CD105+ cells,

• CFU-F BM enriched populations can be cultured with Medium for Human MSC with Stimulatory Supplements for successful propagation of fibroblast-like cells with kinetics documenting ex potential growth after 38 days (median) of culture,

• Cells originated from CFU-F were in contrast to the BM hematopoietic compartment of the recipients origin and were also BCR-ABL negative,

• MSC were not replaced by allogeneic PBPC-graft derived MSC.