ABSTRACT: This study describes the multilineage differentiation pattern of purified CD34+ stem cells obtained from human umbilical cord blood. CD34+ cells were collected from 49 umbilical cord blood samples. Following immunomagnetic purification, cells were double stained with anti CD34 and CD71, CD61, CD7, CD19, CD33, CD36 and triple stained with anti CD34, CD38 and HLA-DR. Analyses were performed using a FACScan flow cytometer. After purification, the mean CD34+ cells purity was 85.49 +/- 7.08%. Several subpopulations of umbilical cord blood CD34+ cells were identified indicating different lineage commitment. The majority of CD34+ cells expressed both CD38 and HLA-DR (91.74 +/- 3.76%), while those lacking CD38 were 3.43 +/- 2.12% (CD38-DR+) and 1.81 +/- 1.54% (CD38-DR-). These data were compared to the expression of lineage commitment markers on purified CD34+ cells from 5 mobilized peripheral blood samples. The percentage of peripheral blood CD34+CD38-DR+ and CD34+CD38-DR- cells was significantly lower than umbilical cord blood, 0.24 +/- 0.18% and 0.04 +/- 0.03% respectively. The knowledge and standardization of umbilical cord blood CD34+ cells phenotype is critical since umbilical cord blood volume is limited. The homogeneity of CD34+ subpopulation phenotype suggests that monitoring of lineage differentiation antigens may not be relevant for clinical use of umbilical cord blood samples. However, the observed higher percentage of pluripotent CD34+38- stem cells in umbilical cord blood compared to peripheral blood, that might explain the successful clinical use of umbilical cord blood even when low number of cells are used, candidates these antigens as the predictive parameter for clinical use of umbilical cord blood samples.     © 1999 Academic Press

Keywords: Umbilical cord blood, CD34+ cells, hematopoietic stem cells, phenotype analysis, immunomagnetic purification.

Reprint requests to: Alberto Degrassi, M.D., Consorzio FENICE, c/o Padiglione Petracco, P. le S. Maria della Misericordia, 33100 Udine, ITALY, phone: +39 0432 547949, fax: +39 0432 548115.

ABSTRACT: Sequencing of HFE exons 2, 3, 4, and 5, and of portions of introns 2, 4, and 5 revealed novel mutations in four of twenty hemochromatosis probands who lacked C282Y homozygosity, C282Y/H63D compound heterozygosity, or H63D homozygosity. Probands 1 and 2 were heterozygous for previously undescribed mutations: exon 2, nt 314T_C (314C; I105T), and exon 2, nt 277G_C (277C; G93R), respectively; these probands were also heterozygous for H63D and C282Y, respectively. Probands 3 and 4 were heterozygous for a previously described but uncommon HFE mutation: exon 2, nt 193A_T (193T; S65C). Proband 3 was also heterozygous for C282Y and had porphyria cutanea tarda, and Proband 4 had hereditary stomatocytosis. Each of these four probands had iron overload. In each proband with an uncommon HFE coding region mutation, I105T, G93R, and S65C occurred on separate chromosomes from those with the C282Y or H63D mutations. Neither I105T, G93R, nor S65C occurred as spontaneous mutations in our probands. The I105T and G93R mutations were linked to haplotypes bearing HLA-A3, -B7 and HLA-A2, -B62, respectively. The S65C mutation was linked to a haplotype characterized by HLA-32. Sixteen other probands did not have an uncommon HFE exon mutation. In 176 normal control subjects, two were heterozygous for S65C, but I105T and G93R were not detected. Nine of twenty probands were heterozygous and two probands were homozygous for a previously described base-pair change at intron 2, nt 3671T_C. One proband without a detectable missense mutation had a previously described intron 5 allele (nt 6700G_A). Heterozygosity for a previously described base-pair change in intron 4 (nt 5636T_C) was detected in all persons we studied who also had the S65C mutation. One proband was heterozygous for a previously undescribed base-pair change at intron 5 (nt 5807A_G). We conclude that uncommon HFE exon and intron mutations may be discovered among hemochromatosis patients who have "atypical" HFE genotypes.    © 1999 Academic Press

Keywords: Hemochromatosis, iron overload, HFE, missense mutation, major histocompatibility region.

Reprint requests to: Dr. James C. Barton, Southern Iron Disorders Center, G-105, 2022 Brookwood Medical Center Drive, Birmingham, Alabama 35209, phone: (205) 877-2888, fax: (205) 877-2039, e-mail: ironmd@dnamail.com.