Possible Mechanisms Accounting for the Growth Factor Independence of Hematopoietic Progenitors From Umbilical Cord Blood zy By zyxwvutsrqp Kurt R. Schibler, Yan Li, Robin K. Ohls, Noel C. Nye, Meghan C. Durham, William White, Kenneth W. Liechty, Trong Le, and Robert D. Christensen Hematopoietic progenitors obtained from the bone marrow of healthy adults fail to undergo clonogenic maturation in vitro if a source of hematopoietic growth factors is not in- cluded in the culture dishes. In contrast, a fraction of simi- larly purified progenitors obtained from umbilical cord blood undergo clonogenic maturation even in the absence of added growth factors. We postulated that production of he- matopoietic growth factors within the culture dishes con- taining theprogenitors of umbilical cord blood origin might be responsible. We postulated further, that thisproduction might be by non-progenitor cells co-plated along with the progenitors, or alternatively by CD34+ cells themselves, or by cells clonally derived from CD34+ cells. To test these pos- sibilities we first assessed the effect of including in the cul- tures neutralizing antibody directed against various growth factors. Inclusion of anti-granulocyte macrophage colony- stimulating factor (GM-CSF) and anti-interleukin-3 (IL-3) (but not anti-IL-2) significantly reduced the growth factor inde- pendence of cord blood progenitors zyxwvuts (P < .005 and P < .01). zyxwvut EMATOPOIETIC PROGENITORS obtained from the blood or liver of early gestation human fetuses, or from umbilical cord blood, have growth factor requirements that are distinct from those of progenitors obtained from adults.”‘ For example, a portion of fetal progenitors undergo clonal maturation in vitro in the absence of added growth factors. Also, a substantial portion of fetal progenitors un- dergo clonogenic maturation in the presence of factors that when used singly are not sufficient to support maturation of progenitors from adults. Examples of factors that as single agents induce clonal maturation of fetal but not adult progen- itors include interleukin-6 (IL-6),’ interleukin-9 (IL-9),3 in- terleukin-l 1 (IL-11); and erythropoietin ( e p ~ ) . ~ , ~ In the latter case, epo alone is sufficient to induce clonal maturation of many of the burst-forming unit-erythroid (BF’U-E) of fetal origin, while BFU-E of adult origin require, in addition to epo, a factor with erythroid burst promoting activity (BPA) such as granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or IL-9. The basis for these differences in growth factor require- ments is not known. We postulated that production of hema- topoietic growth factors within culture dishes that contain the progenitors of umbilical cord blood origin might explain these unique features. We postulated further that the cells producing such growth factors in culture might be either CD34’ cells, progeny of CD34+ cells, or contaminating non- progenitor cells co-plated with the progenitors. Alterna- tively, the unique features might be due to basic differences between the progenitors of adult and fetal origin themselves. Studies were designed to test these hypotheses. H MATERIALS AND METHODS zyxwvutsrq Subjects. Marrow was aspirated from eight healthy adult volun- teers. Blood was obtained from the placental end of the cord by direct needle-puncture of the umbilical vein immediately following 11 healthy vaginal deliveries at term. The studies were performed according to protocols approved by the University of Utah Institu- Blood, Vol 84, zyxwvutsrqp No 11 (December l), 1994: pp 3679-3684 Inclusion of both anti-GM-CSF and anti-IL-3 almost com- pletely ablated the spontaneous colony growth (P < .001). Inclusionof IL-10 also reduced, in a concentration-dependent fashion, the spontaneous generation of umbilical cord blood-derived colonies. Transcripts for GM-CSF and IL-3 were detected, by reverse transcriptase-polymerase chain reaction (RT-PCR), in the CD34+ cells from cord blood and from adult marrow. When plated without added growth fac- tors, however, the CD34+ cells of adult marrow origin failed to produce colonies, whereas 6% of cord blood CD34’ cells similarly cultured did so. When these growth factor indepen- dent colonies were pluckedfrom culture, transcripts for GM- CSF and IL-3 were identified in all. We conclude that pro- duction of GM-CSF and IL-3 occurs within culture dishes containing hematopoietic progenitors of umbilical cord ori- gin, and that this explains some oftheir apparently unique features of in vitro growth. zyxw 0 1994 by The American Society of Hematology. tional Review Board and informed consent was obtained from the participants. Clonogenic cultures. Light density cells (sg < 1.077) were ob- tained by layering marrow or cord blood over Ficoll-Paque (Phar- macia Inc, Piscataway, NJ). Adherent cells were removed by incuba- tion in plastic culture flasks for 90 minutes after which the nonadherent cells were incubated with murine antibodies directed against lymphocytes (anti-Leu-l, anti-Leu-5b. and anti-leu-12; Bec- ton Dickinson, San Jose, CA), monocytes (anti-Leu-MS, Becton Dickinson) and mature erythroid cells (Accurate Scientific and Chemical Corp, Westbury, NY). Antibody-bound cells were re- moved using magnetic beads coated with goat anti-mouse antibodies (Dynal Inc. Great Neck, NY). Cells were plated at densities of either 5 to zyx 10 X lo3 per mL in 1 mL culture dishes or 10 to 20 per mL in 0.1 mL microtiter wells. Cells were plated in a 1% methylcellulose mixture (Sigma Chemi- cals, St Louis, MO) containing 30% fetal calf serum (Hyclone Labo- ratories Inc, Logan, UT), 1% bovine serum albumin (Sigma), 5 X m o m B-mercaptoethanol (Eastman Kodak, Rochester, NY) and alpha MEM (Hyclone). In some experiments, recombinant hu- man epo (Amgen, Thousand Oaks, CA), with a specific activity of From the Division of Human Development and Aging, the Division of Neonatology, the Clinical Research Center, and the Veterans Administration Medical Center, Salt Lake City. UT. Submitted July 15, zyxwvu 1993; accepted August 2, 1994. Supported by Grants HL-44951, HD-01006, HD-00988, and RR- 00064 fromthe National Institutes of Health, a grant from the Pri- mary Children’s Medical Center Foundation, and a Geriatric Re- search Educational and Clinical Center award from the Veterans Administration. Address reprint requests to Kurt R. Schibler, MD,Division of Human Development and Aging, University of Utah School of Medi- cine, 50 N Medical Dr, Salt Lake City, UT 84132. The publication costs of this article weredefrayed zyx in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8411-0014$3.00/0 3679 Downloaded from http://ashpublications.org/blood/article-pdf/84/11/3679/613366/3679.pdf by guest on 18 November 2021