Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

Abstract

Using optimal culture conditions in which the transforming growth factor 11 (TGF-j1) inhibitory loop has been interrupted by antiuense TGF-131 oligonudeotides or antf-TGF-1 serum, we have compared the proliferative capacities and the abUities ofthe CD34+ CD38-ceil populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38-fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38-cells from a typical umbilical cord blood sample produce equivalent numbers of colonyformdngunits (CFU)-gnulocyte/erythrocyte/macrophage/ megakaryocyt, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult tansplantation. In addition, the colonies resulting from the umbical cord blood samples were signiflcantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparatons, as esimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is pardaly compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbiical cord blood is a suitable source of cells for adult transplantation.Small amounts of near-term or neonatal mouse blood has been demonstrated to have the potential to completely reconstitute the hematopoietic system of adult mice (1). This finding has prompted extensive analysis of human umbilical cord blood as a potential source for cells for hematopoietic reconstitution in man (1-4). Various nonmalignant (3, 5) and malignant (7,8) hematopoietic diseases and malignant nonhematopoietic diseases (9, 10) have been successfully treated in children by using umbilical cord blood transplantation. Umbilical cord blood is easily obtainable and therefore could be used to develop banks of HLA-typed cells for general use in adult transplantation ifthe engraftment potential were high enough. Here we report methods for evaluating the relative potentials for engraftment and their use in comparing umbilical cord blood and bone marrow as cellular sources for transplantation.In the past, engraftability of bone marrow samples has largely been analyzed through estimation of the content of colony-forming unit (CFU)-anulocyte/macrophage (GM) (11,13 Purfication of CD34+ Cells. Mononuclear cells were obtained as described (20,23). Hematopoietic progenitors expressing the CD34 antigen were purified using the Applied Immune Sciences (Santa Clara, CA) procedure [soybean agglutinin (SBA) and CD34 CELLector flasks] (20, 24). These cells were then recovered, concentrated, and used for fluorescence-activated cell sorting (FACS), phenotypic anal-

Abrogation of TGF-β activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency

“…[9][10][11]21,26,27 Abrogation of autocrine or paracrine TGF-␤ with a neutralizing antibody or specific antisense oligonucleotides can improve the viability and proliferative potential of ex vivo cultured primitive hematopoietic cells. 9,11,21,[26][27][28][29] We have previously shown that true murine repopulating stem cell activity as measured by the quantitative murine competitive repopulation assay is also increased two-to four-fold by inclusion of neutralizing TGF-␤ antibody during culture in IL-3, IL-6 and SCF. 12 A number of potential mechanisms have been suggested for these inhibitory actions.…”

“…Other investigators have also reported that abrogation of autocrine/paracrine TGF-␤ has beneficial effects on viability and proliferation of primitive hematopoietic cells. 8,[13][14][15][16] Diminution of TGF-␤ activity during ex vivo culture by neutralizing antibodies or antisense oligonucleotides has been shown to increase retroviral gene transfer efficiency into committed progenitor cells such as CFU-GEMM (colony-forming unit granulocyte-erythroid-megakaryocyte-macrophage). 17 However, assessment of gene transfer into committed progenitor cells is often not predictive of the efficiency of gene transfer into true repopulating stem cells.…”

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

“…Human umbilical cord blood, which contains hematopoietic stem/progenitor cells at a frequency equal to or greater than that of adult bone marrow (28,29), and with a high quality for proliferation (30)(31)(32)(33)(34) and self-renewal capacity, as estimated by replating colonies in vitro (30,31,35), has shown promise clinically as an alternative source of human transplantable and marrow-repopulating cells (36)(37)(38)(39)(40)(41)(42)(43). Hematopoietic stem and progenitor calls in human cord blood may be ideal targets as recipients of potential gene therapy for hematopoietic disorders (44).…”

The high proliferative potential-quiescent (HPP-Q) cell assay allows an optimized evaluation of gene transfer efficiency into primitive hematopoietic stem/progenitor cells

“…They are CD34 + /CD38 − , /Kit low , /Trf-R low , /FLT3 low , /Mpl low and /IL6-R low . 6,7,22 Interestingly, when CD34 + /Kit − , CD34 + /Kit low and CD34 + /Kit high cells were injected separately into preimmune fetal sheep recipients, only the CD34 + human marrow cells that express low levels of Kit protein, corresponding to the HPP-Q cells allowed a long-term engraftment. 24 Several in vivo models have been developed to test the ability of human stem cells to repopulate immune-deficient mice such as the NOD/SCID mice.…”

“…They provide larger mixed or GM colonies containing various hematopoietic lineages. [5][6][7] They respond to the inhibitory effect of very low endogenous concentrations of TGF-␤1. 10 In this study, we consider only the HPP-Q-Mix as they are the most primitive and could belong to the stem cell compartment.…”