Marie-Noëlle Monier

Release from Quiescence of Primitive Human Hematopoietic Stem/Progenitor Cells by Blocking Their Cell-Surface TGF-β Type II Receptor in a Short-Term In Vitro Assay

Genetic alterations of the signaling cascade of transforming growth factor‐β (TGF‐β) are often associated with neoplastic transformation of primitive cells. This demonstrates the key role for this pleiotropic factor in the control of quiescence and cell proliferation in vivo. In the high proliferative potential‐quiescent cell (HPP‐Q) in vitro assay, the use of TGF‐β1 blocking antibodies (anti‐TGF‐β1) allows the detection within two to three weeks of primitive hematopoietic cells called HPP‐Q, which otherwise would not grow. However, the possibility of triggering cell proliferation by blocking the cell‐surface TGF‐β receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF‐βRII (anti‐TGF‐βRII) on CD34+CD38− hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti‐TGF‐β1. About twice as many HPP colony‐forming cells were detected in the presence of anti‐TGF‐β1 or anti‐TGF‐βRII, compared to the control (p < 0.02). Moreover, anti‐TGF‐βRII was as efficient as anti‐TGF‐β1 for activating multipotent HPP‐granulocyte erythroid macrophage megakaryocyte and HPP‐Mix, bipotent HPP‐granulocyte‐macrophage (GM) and unipotent HPP‐G, HPP‐M and HPP‐BFU‐E. We therefore propose the use of anti‐TGF‐βRII to release primitive cells from quiescence in the HPP‐Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF‐β1 may be a pleiotropic regulator of somatic stem cell quiescence.

Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-β1 concentrations

We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-β1 (TGF-β1) exerts a pleiotropic effect. TGF-β1 confers to these slow-dividing cells a mitogenic receptorlow phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-β1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 107-108, increasing to 1012-1013 in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-β1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 1016-1017 (∼60 population doublings, ∼4×1018 keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in α6 and β1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-β1, which is to promote undifferentiated keratinocyte amplification.

p21cip1 mRNA is controlled by endogenous transforming growth factor-?1 in quiescent human hematopoietic stem/progenitor cells

Transforming growth factor‐β1 (TGF‐β1) has been described as an efficient growth inhibitor that maintains the CD34+ hematopoietic progenitor cells in quiescence. The concept of high proliferative potential‐quiescent cells or HPP‐Q cells has been introduced as a working model to study the effect of TGF‐β1 in maintaining the reversible quiescence of the more primitive hematopoietic stem cell compartment. HPP‐Q cells are primitive quiescent stem/progenitor cells on which TGF‐β1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF‐β1 with a TGF‐β1 blocking antibody or a TGF‐β1 antisense oligonucleotide. In nonhematopoietic systems, TGF‐β1 cooperates with the cyclin‐dependent kinase inhibitor, p21cip1, to induce cell cycle arrest. We therefore analyzed whether endogenous TGF‐β1 controls the expression of the p21cip1 in the CD34+ undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti‐TGF‐β1 is followed by a rapid decrease in the level of p21cip1 mRNA whereas TGF‐β1 enhances p21cip1 mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21cip1 in the cell cycle control of early human hematopoietic quiescent stem/progenitors and not only in the differentiation of more mature myeloid cells as previously described. The modulation of p21cip1 observed in response to TGF‐β1 allows us to further precise the working model of high proliferative potential‐quiescent cells. J. Cell. Physiol. 184:80–85, 2000.

Control of hematopoietic stem/progenitor cell fate by transforming growth factor-β

A major obstacle to the use of adult somatic stem cells for cell therapy is our current inability to fully exploit stem cell self-renewal properties. The challenge is to obtain defined culture systems where cycling of primitive stem/progenitor cells is stimulated, while their differentiation and senescence are prevented. The cytokine transforming growth factor-beta1 (TGF-beta1) appears as a potential regulator of hematopoietic stem/ progenitor cell self-renewal, as it participates in the control of cell proliferation, survival/apoptosis, and cell immaturity/differentiation. TGF-beta1 acts via a complex regulatory network involving intracellular signaling molecules and cell surface receptors. According to the High Proliferative Potential-Quiescent (HPP-Q) cell working model that we introduced previously, TGF-beta1 maintains primitive hematopoietic stem/progenitor cells in a quiescent or slow cycling state, in part by downmodulating the cell surface expression of mitogenic cytokine receptors, thus preventing cells from responding rapidly to a mitogenic signal. We have established that this modulation concerns the tyrosine kinase receptors KIT and FLT3, and the IL-6 receptor (IL-6R), three important cytokine receptors controlling early human hematopoietic stem/progenitor cell development. In this article. we show a similar modulation by TGF-beta1 of a fourth receptor: the TPO receptor (MPL). As a consequence, TGF-beta1 decreased the cell cycle entry of stem/progenitor cells stimulated by the respective ligands of these receptors, the cytokines SF, FL, IL-6, and TPO, whereas neutralization of TGF-beta1 increased the cell responsiveness to these mitogenic cytokines. Other aspects of the function of TGF-beta1 in the regulation of early hematopoiesis (i.e., the control of stem/progenitor cell survival and immaturity) are reviewed in the discussion.

Use of Xenofree Matrices and Molecularly-Defined Media to Control Human Embryonic Stem Cell Pluripotency: Effect of Low Physiological TGF-β Concentrations

To monitor human embryonic stem cell (hESC) self-renewal without differentiation, we used quantitative RT-PCR to study a selection of hESC genes, including markers for self-renewal, commitment/differentiation, and members of the TGF-beta superfamily and DAN gene family. Indeed, low commitment/differentiation gene expression, together with a significant self-renewal gene expres sion, provides a better pluripotency index than self-renewal genes alone. We demonstrate that matrices derived from human mesenchymal stem cells (hMSCs) can advantageously replace murine embryonic fibroblasts (MEF) or hMSC feeders. Moreover, a xenofree molecularly-defined SBX medium, containing a synthetic lipid carrier instead of albumin, can replace SR medium. The number of selected differentiation genes expressed by hESCs in these culture conditions was significantly lower than those expressed on MEF feeders in SR medium. In SBX, the positive effect of a non-physiological concentration of activin A (10-30 ng/mL) to reduce differentiation during self-renewal could also be obtained by physiological concentrations of TGF-beta(100-300 pg/mL). In contrast, these TGF-beta concentrations added to activin favored differentiation as previously observed with TGF-beta concentrations of 1 ng/mL or more. Compared to SR-containing medium, SBX medium promoted down-regulation of CER1 and LEFTIES and up-regulation of GREM1. Thus these genes better control self-renewal and pluripotency and prevent differentiation. A strategy is proposed to analyze, in more physiological, xenofree, molecularly-defined media and matrices, the numerous genes with still unknown functions controlling hESCs or human-induced pluripotent stem cells (iPS).