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.
"This information could be used by cord blood banks to reduce costs and optimize resources by carefully selecting donors . As cord blood banking is expensive , strategies such as these may allow banks to optimally choose donors and therefore be able to bank the most suitable cords for transplant ( Kogler et al , 1996 ; Fraser et al , 1998 ; Querol et al , 1998 ; Ballen et al , 2000b ) . Our study looked only at engraftment of cord blood from full term ( . "
[Show abstract][Hide abstract] ABSTRACT: Umbilical cord blood is an alternative stem cell source for patients without matched family donors. In this study, we examined several parameters that have not been studied in detail -- radiation dose, cell dose, age of mice, and maternal and neonatal characteristics of the cord blood donor -- that affect engraftment of cord blood in non-obese diabetic-severe combined immunodeficient (NOD--scid) mice. Engraftment, measured using flow cytometry analyses of human CD45(+) cells, was highest in 400 cGy-treated mice. Successful engraftment was demonstrated up to 6 months, with a mean engraftment of 31% (range 0--67%) of human cells in recipient bone marrow. Engraftment was skewed to B lymphocytes. The radiation dose of 350 cGy resulted in superior survival of the murine recipients compared with 400 cGy (P = 0.03). The sex of the NOD--scid recipients had a significant effect on survival (female superior to male, P = 0.01), but not on engraftment. There were high levels of variability among different cord units and among animals injected with the same cord unit. This variability may limit the clinical usefulness of the NOD--scid mice as hosts for the quantification of human stem cells.
British Journal of Haematology 08/2001; 114(1):211-8. DOI:10.1046/j.1365-2141.2001.02904.x · 4.71 Impact Factor
"banks, mainly for related transplants (n 13). CB units were collected, processed and thawed following previously published procedures (Rubinstein et al, 1995; Ko Ègler et al, 1996). The method of counting the number of granulocyte±macrophage colony-forming units (CFU-GM) and CD34 1 cells collected and infused varied considerably between centres and was therefore not included in the analysis. "
[Show abstract][Hide abstract] ABSTRACT: Immune recovery after cord blood transplantation (CBT) is of concern owing to the low number of lymphocytes transferred with the graft and their immaturity. Risk factors influencing lymphocyte subset reconstitution related to disease, patient, donor and transplant were studied in 63 children (< 16 years), given either related (n = 14) or unrelated (n = 49) CBT for malignant (n = 33) or non-malignant diseases (n = 30). Only children with sustained myeloid engraftment were analysed. Absolute numbers of T (CD3(+), CD4(+), CD8(+)), B and natural killer (NK) cells were reported 2--3, 6, 9, 12 and 12--24 months after CBT. Median patient age was 4.0 years (0--15) and median follow-up was 23 months (1.7--61.0). Twenty-six patients received human leucocyte antigen (HLA)-matched CBT and 37 received HLA-mismatched CBT. The median number of nucleated cells (NCs) collected/recipient weight was 6.1 x 10(7)/kg. In this selected population, the estimate 2 year survival was 85%. Lymphocyte reconstitution (defined as the median time to reach the normal value of age-matched healthy children) was 3, 6 and 8 months for NK, B and CD8(+) cells, while it was 11.7 months for both CD3(+) and CD4(+) lymphocytes. In the multivariate analysis, factors favouring T-cell recovery were: related donor (P = 0.002); higher NCs/kg (P = 0.005) and recipient cytomegalovirus (CMV)-positive serology (P = 0.04). Presence of acute graft-versus-host disease (GVHD) delayed T-cell recovery (P = 0.04). To summarize, in children with sustained myeloid engraftment the concern that lymphocyte recovery after CBT could be delayed does not appear to be substantiated by our results.
British Journal of Haematology 08/2001; 114(1):42-8. DOI:10.1046/j.1365-2141.2001.02900.x · 4.71 Impact Factor
"Since the developmental age of hematopoietic progenitors is a factor in their potential to form colonies in vitro, a comparison between the numbers of FBM CFU-C and UCB CFU-C is likely to be the most relevant in estimating the potential of FBM cells to engraft. The number of myeloid and mixed-lineage CFU-C detected from a single UCB harvest have been reported to range from 5 x 10 5 to 7 x 10 5 (Kletzel et al, 1997; Kögler et al, 1996). Thus, the number of CFU-C available from a single FBM harvest can be as much as 12-fold greater than from UCB. "
[Show abstract][Hide abstract] ABSTRACT: We examined the potential of human fetal bone marrow (FBM) as a source of haematopoietic stem cells for transplantation. The median number of cells obtained between 20 and 24 weeks' gestation was 1.9 x 109 and a median 1.17 x 108 of these cells expressed CD34. Flow cytometry was also used to estimate the content of three different candidate stem cell populations in the tissues older than 20 weeks' gestation. A median 8.8 x 105 CD34++CD38- cells, 1.37 x 106 CD34++CD4+ cells and 2.20 x 106 CD34++CD90+ cells were detected. The content of colony-forming units culture (CFU-C) in the FBM ranged from 2.8 x 104 to 6.0 x 106 per fetus. The CFU-C content could be expanded 50-fold by culture for 1 week in serum-deprived medium and the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. Positive selection of FBM CD34+/++ cells was achieved using the Baxter Isolex 50 device. An average purity of 82% and yield of up to 19% of CD34+/++ cells was achieved. T cells were depleted by 99.84%. Analysis of candidate stem cell populations and primitive CFU-C suggested a preferential enrichment of these cells over the total population of CD34+/++ cells. All FBM samples were found to be free of microbial contamination at the time of harvest and after selection of CD34+/++ cells. Thus, FBM is a safe source of stem cells. The large number of progenitors and candidate stem cells that can be obtained from FBM makes it suitable for in utero and possibly postnatal transplantation.
British Journal of Haematology 05/2000; 109(1):173-81. DOI:10.1046/j.1365-2141.2000.02009.x · 4.71 Impact Factor
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