Antoinette Hatzfeld

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.

Mitogenic properties of major extracellular proteins

The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

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.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1low, BMPR1Blow, FLT4low, LRRC32low, and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

A sub-population of high proliferative potential-quiescent human mesenchymal stem cells is under the reversible control of interferon α / β

Type I interferon (IFN) is shown to control the reversible quiescence of a primitive human bone marrow mesenchymal stem cell (MSC) subpopulation. A 24 h pre-treatment of Stro1+/GlycoA- or CD45-/GlycoA- subpopulations with a monoclonal antibody (mAb) against the IFNAR1 chain of the human type I IFN receptor (64G12), or with a polyclonal anti-IFNα antibody, resulted in a marked increase in the number of very large colonies (CFU-F >3000 cells) obtained in the presence of low, but necessary, concentrations of bFGF. Over a 2-month culture period, this short activation promoted a faster and greater amplification of mesenchymal progenitors for adipocytes and osteoblasts. Activation correlated with inhibition of STAT1 and STAT2 phosphorylation and of STAT1 nuclear translocation. A non-neutralizing anti-IFNAR1 mAb was ineffective. We demonstrate that control and activated MSCs express ST3GAL3, a sialyltransferase necessary to produce the embryonic antigens SSEA-3 and -4. Interestingly, activated MSC progeny expressed SSEA-3 and -4 at a higher level than control cultures, but this was not correlated with a significant expression of other embryonic markers. As MSCs represent an essential tool in tissue regeneration, the use of 64G12, which rapidly recruits a higher number of primitive cells, might increase amplification safety for cell therapy.

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).

Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells: Possible role of ABC plasma membrane transporters in maintaining human stem cell pluripotency

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporterlow phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.

Le C3 stimule la prolifération des cellules humaines pré-B de la lignée Raji

In a defined medium in which transferrin (3 micrograms/ml) was the only source of exogenous proteins, Raji cells of the human pre-B lymphoblastoid cell line died within 48 h after forming polykaryons. The simple addition of purified C3 at a concentration equal to or higher than 3 micrograms/ml allowed Raji cells to divide. This preliminary report provides a defined system for studying the mitogenic effect of human C3 or C3 fragments upon proliferation of human B-cells lines.