Autocrine transforming growth factor β1 blocks colony formation and progenitor cell generation by hemopoietic stem cells stimulated with steel factor

Abstract

The ability of Steel Factor (SF) to stimulate colony formation and progenitor cell generation by hemopoietic stem cells (HSCs) in vitro in the absence of interleukin 3 (IL-3) was investigated. IL-3 was required for HSC proliferation, and no or restricted proliferation occurred in the presence of SF, IL-6, IL-11, or IL-12 as single factors or in combination. Neutralizing concentrations of anti-transforming growth factor (TGF)-beta 1 antibodies enhanced progenitor cell generation 2-3-fold in the presence of IL-3, but 75 to over 300-fold when cultures contained at least SF in the absence of IL-3. Exogenous TGF-beta 1 fully abrogated the antibody effects. In the presence of antibodies to TGF-beta 1, SF alone stimulated the delayed formation of small blast cell colonies and SF synergized with IL-6, IL-11, or IL-12 to greatly hasten colony formation, enhance colony number and size, and increase colony forming unit-culture (CFU-C) output from suspension cultures of enriched HSC populations. Secondary CFU-C colonies were significantly larger when IL-3 was absent during the suspension culture phase. Single cell and limiting dilution analysis using a homogenous colony forming unit-spleen (CFU-S) day-12 population and an 800-fold enriched long-term repopulating HSC fraction, respectively, indicated that TGF-beta 1 was an autocrine product of these HSC subsets. Addition of nucleosides, insulin, extra glucose, or serum could not replace the effects of the anti-TGF-beta 1 antibody. While these data offer one possible explanation for reports on the inability of SF to stimulate HSC proliferation, they present the basis for a novel model of the regulation of HSC activation wherein: 1) close-range interactions of HSCs with mesenchymal stromal cells do not exclusively determine maintenance of HSC quiescence; 2) competence acquisition by dormant HSCs may involve the down-regulation or inactivation of autocrine TGF-beta 1; and 3) SF may act as a primary growth factor rather than exclusively as a synergistic cytokine.

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

“…4 During retroviral gene transfer, there are a number of potential sources of deleterious TGF-␤, including autocrine production by primitive target cells, paracrine production by other marrow hematopoietic elements such as stromal cells, or release of TGF-␤ by retroviral producer cell lines into supernatants used for transduction. 9,11,38 Increased retroviral gene transfer efficiency into committed progenitor cells after abrogation of TGF-␤ activity with antisense oligonucleotides or a neutralizing antibody has been previously reported. 17,38 Our report is the first to document improved retroviral gene transfer into long-term repopulating cells.…”

“…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.…”

Stimulation of Adult Human Bone Marrow by Factors Secreted by Fetal Liver Hematopoietic Cells: In Vitro Evaluation Using Semisolid Clonal Assay System

“…The observation that TGF-β1 was consistently associated with hematopoietically active tissues had clear implications of its involvement in hematopoiesis. It has even been observed that TGF-β1 could be an activator [36][37][38] or inhibitor [32,33,[39][40][41][42][43][44][45] of hematopoiesis, depending on the target population. The activatory role of TGF-β1 in these experiments was confined only to GM progenitors and required that higher concentrations of TGF-β1 (ng/ml) be applied.…”

Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures

“…Previously we have found these cytokine combinations to synergize in progenitor cell generation and colony formation of murine bone marrow cells, 12,13 and to be extremely useful for detecting the presence of very low concentrations of TGF-␤1 (ie down to 1 pg/ml). 14,15 From Figure 1 it appears that BMP-9 concentrations of 100 ng and higher, completely abrogated CFU-C generation stimulated by SF+IL-12, and almost completely when low concentrations of IL-3 (5 U) were included. The same effect could be observed if IL-12 was replaced by IL-11 (data not shown).…”