Release of early human hematopoietic progenitors from quiescence by antisense transforming growth factor beta 1 or Rb oligonucleotides.

Abstract

SummaryWe have used antisense oligonudeotides to study the roles of transforming growth factor fl (TGF-fl) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-3 sequence significantly enhanced the frequency of colony formation by multi-lineage, early erythroid, and granulomonocytic progenitors, but did not affect colony formation by late progenitors. Single cell culture and limiting dilution analysis indicated that autocrine TGF-fl is produced by a subpopulation of early progenitors. Antisense Rb but not antisense p53 yielded similar results in releasing multipotential progenitors (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte) from quiescence. Rb antisense could partially reverse the inhibitory effect of exogenous TGF-fl. Anti-TGF-fl blocking antibodies, antisense TGF-3, or Rb oligonucleotides all had similar effects. No additive effects were observed when these reagents were combined, suggesting a common pathway of action. Our results are consistent with the model that autocrine production of TGF-fl negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

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

“…25 TGF-␤ are produced by marrow microenvironmental components such as stromal cells and macrophages, as well as by primitive hematopoietic progenitors themselves. [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.…”

“…7 These cytokines are produced by marrow microenvironmental elements and have paracrine effects on hematopoietic cells, and have also been shown to be produced in an autocrine manner by primitive hematopoietic cells themselves. [8][9][10][11] Our previous studies have shown that maintenance of murine long-term repopulating stem cells during ex vivo culture is affected by modulation of transforming growth factor-␤ activity. 12 The presence of TGF-␤1 during ex vivo culture had significant suppressive effects on both short and long-term repopulating activities and the addition of anti-TGF-␤ neutralizing antibody improved murine stem cell repopulating activity.…”

“…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.…”

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

“…Blocking the Rb gene or CDKI such as p27 by antisense oligonucleotides has similar effects. 5,9 We have observed that the earlier high proliferative potential-mixed (HPP-Mix) are those which are inhibited by the lowest concentrations of TGF-␤1 (10 pg/ml), whereas later HPP-GM are only sensitive to concentrations between 100 and 300 pg/ml. 10 Still later pro-TGF-␤1 could induce reversion to quiescence of the more primitive CD34 + cells transduced after one cell cycle.…”

“…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.…”