Hematopoietic Transplant Potential of Unrelated Cord Blood: Critical Issues

ArticleinJournal of Hematotherapy 5(2):105-16 · May 1996with5 Reads
DOI: 10.1089/scd.1.1996.5.105 · Source: PubMed

    Abstract

    To date, hematopoietic stem and progenitor cells from human umbilical cord blood (CB) have been employed in approximately 90 allogeneic (56 sibling and 34 unrelated) matched and mismatched transplantations worldwide with easy and successful restoration of hematopoiesis. Requests for stem cell preparations from CB will continue to increase. Thus, as a pilot study, the examination and standardization of unrelated cord blood-derived stem cell preparations and banking as well as their biologic characterization were initiated. Up to October 1995, a total of 574 samples [mean volume 79 +/- 26 ml, total nucleated cells (NC) 8.5 +/- 5 x 10(8), BFU-E 9.5 +/- 8.6 x 10(5), CFU-GM 5.7 +/- 6.3 x 10(5), CFU-GEMM 1.6 +/- 1.9 x 10(5)] from cord-derived or placental-derived residual blood have been defined by hematologic, immunologic, and microbiologic criteria. These CB samples were collected from the umbilical cord vein immediately after vaginal full-term delivery (n = 450) or cesarean section (n = 124) and stored frozen in liquid nitrogen. Seven percent of all samples collected could not be considered for potential transplants because of volumes < 40 ml. Only 5.0 ml of a CB sample is required for routine laboratory testing, consisting of HLA class I typing, HLA class II typing by sequence-specific oligonucleotide probes (PCR-SSOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony-forming and LTC-IC assays, and CD34+ status. To assess the potential problem of contaminating maternal cells, a PCR was performed on 7 representative samples. During the initial 6 months of the unrelated CB collection program, a median bacterial contamination rate of 18% (20% skin flora species, 80% perineal flora species) was encountered, which has since been reduced to < 1% through practical experience. With regard to viral infections, maternal sera was tested for HBsAg (0.6% positive), anti-HCV (0%), anti-HAV (IgG 18%, IgM 0%), anti-HIV-1-2 (0%), anti-EBV (IgG 98%, IgM 0%), anti-HTLVI-II (0%), anti-CMV (IgG 43%, IgM 0.4%), toxoplasmosis (46%) and syphilis (0%). In addition, all cord blood samples were tested by PCR for CMV infection. With regard to its clinical relevance, it is important that only 0.3% of all the samples were positive for CMV by this sensitive method. This may represent a critical advantage of CB grafts over bone marrow (BM) since, in contrast, > 40% of the unrelated BM donors have been identified to be positive for CMV. An additional advantage of CB is that since 20% of CB samples were collected from ethnic minorities, it appears possible to balance common HLA types and uncommon HLA types represented in this group. In summary, with the extensive practical experience of the obstetric collection team as well as the stem cell-processing laboratory, it appears feasible to obtain a 90% yield of unrelated CB-derived stem cell preparations for banking, which clearly should meet the medical and regulatory qualification criteria required for clinical transplantation. To test the feasibility of hematopoietic transplant potential of unrelated CB for adult patients, ex vivo expansion of CD34+-enriched stem/progenitor cell populations isolated from fresh or frozen CB was attempted in the presence of rh-IL-3, rh-IL-6, rh-EPO, rh-GM-CSF, and rh-SCF with or without fit 3. At varying time points (days 0, 2, 4, 7, 14, 21), the contents of these cultures were analyzed for the numbers of cells, CFC (BFU-E, CFU-GM, CFU-GEMM), and LTC-IC. In this setting, the increase of cells was 200-fold, that of CFC 70-fold, and most importantly that of LTC-IC was 4.5-fold after 7 days in culture in the presence of flt3. In conclusion, LTC-IC derived from CB can be maintained and considerably expanded ex vivo from highly enriched CD34 + CB cell populations from fresh or frozen CB samples.