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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).

Here we present a simple two-step in vitro model of vascularized trophoblastic tissue derived from human embryonic stem (hES) cells. The first step is the formation of cystic embryoid bodies (EBs) in suspension in a semisolid methyl cellulose medium, within which an endothelial platelet/endothelial cell adhesion molecule-1 (PECAM-1(+)) cell network develops. In a second step, deposition of these EBs on the bottom of nontreated, polystyrene tissue culture plates, leads by centrifugal outgrowth of the EB to the emergence of an adherent cell layer, with which a PECAM-1(+) network is associated. Cells of this adherent layer expressed VE-cadherin (CD144), PECAM-1 (CD31), and alpha-fetoprotein (alpha-FP). Trophoblastic differentiation was strongly suggested by the secretion of beta-human chorionic gonadotropin (beta-hCG) and by the presence of the cytotrophoblast and syncytiotrophoblast marker GB25. The INSL4 gene, a cyto and syncytio-trophoblast marker, was also highly expressed in the adherent layer, as well as other trophoblast genes such as CGA, CDX1, CDX2, and HAND1, compared to hES cell gene expression taken as reference. In contrast, expression of self-renewal genes, such as TERT, POU5F1, ZFP42, GDF3, and NODAL were decreased. No ectodermal or endodermal genes were expressed, but the mesodermal genes PECAM-1 and GATA2 were. The possibility of removing the EBs during the second step would permit analysis of their relative contribution to angiogenesis or possible hemangioblast formation, compared to that of the trophoblastic adherent layer. This primitive vascularized trophoblastic model could also provide a tool to study early steps of normal and pathological placental development.

Human embryonic stem cells (hESCs) are derived from pre-implantation embryos given to research with the informed consent of the parents. These cells cannot give rise to a human being: they are not totipotent. They have an unlimited self-renewal capacity and they can generate the three embryonic germ layers, their respective derivatives and the extra-embryonic tissues: they are pluripotent. These cells represent an outstanding material for understanding functional genomics of not yet characterized human genes. They will be an important tool for pharmaceutical and clinical research.

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. © 2000 Wiley-Liss, Inc.

Genetic alterations of the signaling cascade of transforming growth factor-beta (TGF-beta) 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-beta1 blocking antibodies (anti-TGF-beta1) 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-beta receptors has not been investigated until now. We have tested here the efficiency of a blocking antibody against TGF-betaRII (anti-TGF-betaRII) on CD34(+)CD38(-) hematopoietic cells, a subpopulation enriched in primitive stem/progenitor cells, and compared its effect with that of anti-TGF-beta1. About twice as many HPP colony-forming cells were detected in the presence of anti-TGF-beta1 or anti-TGF-betaRII, compared to the control (p < 0.02). Moreover, anti-TGF-betaRII was as efficient as anti-TGF-beta1 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-betaRII to release primitive cells from quiescence in the HPP-Q assay. This strategy could be extended to nonhematopoietic tissues, as TGF-beta1 may be a pleiotropic regulator of somatic stem cell quiescence.

Somatic stem cells are largely quiescent in spite of their considerable proliferative potential. Transforming growth factor-(beta)1 (TGF-(beta)1) appears to be a good candidate for controlling this quiescence. Indeed, various mutations in the TGF-beta signalling pathway are responsible for neoplasic proliferation of primitive stem/progenitor cells in human tissues of various origins. In hemopoietic single cell culture assays, blocking autocrine and endogeneous TGF-(beta)1 triggers the cell cycling of high proliferative potential undifferenciated stem/progenitor cells. However, it has never been demonstrated whether TGF-(beta)1 has an apoptotic effect or a differentiating effect on these primitive cells, as already described for more mature cells. Using single cell experiments both in liquid or semi-solid culture assays and dye tracking experiments by flow cytometry, we demonstrate that low, physiological concentrations of TGF-(beta)1, which specifically maintain primitive human hemopoietic stem/progenitor cells in quiescence, have a reversible effect and do not induce apoptosis. We moreover demonstrate that these low concentrations prevent the rapid loss of the mucin-like protein CD34, a most common marker of immature hematopoietic stem/progenitor cells, which is progressively lost during differentiation. TGF-(beta)1 not only up-modulated the CD34 antigen before S phase entry but also maintained a high level of CD34 expression on cells which had escaped cell cycle inhibition, suggesting that proliferation inhibition and differentiation control by TGF-(beta)1 may be independent. These data provide additional evidence that TGF-(beta)1 acts as a key physiological factor ensuring the maintenance of a stem cell reserve.

It has been previously demonstrated that prestimulation with cytokines could improve gene transfer in hematopoietic progenitors. However, we have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless an autocrine transforming growth factor-beta 1 (TGF-beta 1) is blocked by specific oligonucleotide antisense or antiserum (Hatzfeld et al., 1991, J. Exp. Med., 174, 925). We now report that a 10-hr cytokine prestimulation of SBA-CD34high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 1% to 23.8% and addition of anti-TGF-beta serum doubles this increase (47.3%). Interestingly, the effect of anti-TGF-beta preincubation on gene transfer is most effective on the most immature progenitors, which develop into high proliferative potential mixed colonies with 1-2 x 10(5) cells. Anti-TGF-beta serum pretreatment increases gene transfer in these early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) from 54.1% to 93.3%. It augments significantly the stability of gene expression in all subpopulations of mixed colonies. Colonies obtained after pretreatment with anti-TGF-beta serum are larger and the expression of the stably integrated recombinant provirus does not reduce their size. This prestimulation method provides a substantial improvement for gene transfer efficiency within the quiescent stem cell compartment that is responsible for long-term engraftment.

The effect of human fibrinogen on the proliferation of purified SBA- CD34+ human bone marrow progenitors was investigated in clonal cultures. Fibrinogen alone or in combination with erythropoietin had no significant effect. However, in the presence of recombinant human interleukin-3 (IL-3), fibrinogen increased significantly in a dose- dependent manner the number of mixed and burst-forming unit-ethrocyte-- derived colonies, whereas the number of other colonies did not significantly change. In the presence of fibrinogen, low concentrations of IL-3 (0.17 U/mL) produced three times more mixed colonies than without fibrinogen, reaching the number of colonies obtained with optimal concentrations of IL-3 (1.67 U/mL). Fibrinogen fragment D had the same effect in the presence of IL-3 as intact fibrinogen, whereas fibrinogen fragment E and human collagen IV did not. This effect was not mediated by integrins, because peptides or monoclonal antibodies that block fibrinogen binding on integrins alpha IIb beta 3, alpha v beta 3 (RGD-peptides), alpha m beta 2 (OKM-1), and alpha x beta 2 (HC1/1) did not affect the observed mitogenic effect. The mitogenic effect of fibrinogen and its D fragment was not mediated by induction of IL-6 or granulocyte--colony-stimulating factor secretion, because it was not inhibited by blocking antisera against these two growth factors. Our results indicate that fibrinogen potentiates the effect of IL-3 on primitive hematopoietic progenitors and suggest that the mitogenic effect of fibrinogen could be mediated via a specific mitogenic receptor that does not belong to the integrin family.

We have previously shown (Zhou et al: Blood, 72:1870, 1988) that IL3, added with low concentrations of CSF-1 (1 ng/ml) to normal human CD34+ enriched cells, promoted the development of various types of colonies including those containing immature monocytes. However, when high concentrations of CSF-1 (20 ng/ml) were added alone or together with IL3, smaller colonies with mature macrophages were found. Here we show by in situ hybridization that IL3 allows the development, from CD34+ cells, of a subpopulation of immature progenitors which express the CSF-1 receptor (c-fms) mRNA. The expression of c-FMS protein was also substantiated by immunocytochemical studies using anti-c-fms antibody. The percentage of c-fms positive cells peaked at day 7 and began to decrease thereafter. When anti-CSF-1 antibodies were included in the culture, the decrease in c-fms mRNA after day 7 was abrogated. This indicated that endogenous CSF-1 was produced as CD34+ cells developed into monocytes or progenitors of monocytes and that CSF-1 modulates c-fms expression. We further demonstrated that when a high dose of CSF-1 (20 ng/ml) was added at day 7 to IL3-stimulated CD34+ cells, a rapid down-regulation of c-fms mRNA and protein was seen. No down-regulation was observed with low concentration of CSF-1 (1 ng/ml). The possibility that different concentrations of CSF-1 could modulate the development of monocytic progenitors is discussed.

Human umbilical cord blood CD34+ cells were purified using a two-step procedure by elimination of the soybean agglutinin-binding cells and by a positive panning selection with a CD34 monoclonal antibody. The isolated fraction was 88-97% pure CD34+ cells. A yield of 48.5% was obtained when comparing the number of cells recovered in the CD34(+)-purified fraction and the number of CD34+ cells detected in the initial mononuclear cell fraction. By flow cytometry, we observed that the CD34+ cells that were not recovered were those that had the lower expression of CD34 antigen and were therefore the more mature cells. A high recovery of CFU-GEMM progenitors (73.9%) was also observed. These data suggest the possibility of purifying CD34+ umbilical cord blood cells for clinical applications, in particular for umbilical cord blood banking.

We describe in this chapter the development of a xenofree molecularly defined medium, SBX, associated with xenofree matrices, to maintain human embryonic stem cell (hESC) pluripotency as determined by phenotypic, functional and TLDA studies. This simple, inexpensive, and more physiological culture condition has been chosen because (1) it is xenofree and molecularly defined; it is devoid of albumin, which is a carrier of undefined molecules; (2) it maintains pluripotency, but very significantly reduces differentiation gene expression during hESC self-renewal, as compared to the widely used culture conditions tested so far; and (3) it can be further improved by replacing high concentrations of expensive additives by physiological concentrations of new factors. Xenofree molecularly defined media and matrices represent valuable tools for elucidating still unknown functions of numerous embryonic genes using more physiological culture conditions. These genes encode potential new factors controlling hESC self-renewal and pluripotency.

4 ng/ml bFGF is indispensable for hESC cultured on mouse embryonic fibroblasts (MEF), withdrawal of bFGF drives the hESC to differentiate. In order to exploit effect of bFGF on MEF, we collected a series of MEF conditioned medium (bFGF-MCM) by co-culturing MEF with increasing bFGF concentrations: 0.03, 0.1, 0.3, 1 and 4 ng/ml. The primitivity of hESC cultured in bFGF-MCM was estimated by morphology and alkaline phosphatase staining. Compared with the control medium (medium conditioned without bFGF: MCM), percentage of undifferentiated colony was increased from 23% to 29%, 44%, 74%, 77% and 78%, respectively. However, percentage of undifferentiated colony in the blank medium (medium conditioned with bFGF but without MEF: bFGF-SR) was from 13% to 31%. This indicated that low concentration of bFGF acted on MEF and stimulated MEF producing effective conditioned medium for maintaining hESC. To identify active elements in the effective conditioned medium can help to understand mechanisms of hESC self-renewal.

We describe in this chapter the development of a xenofree molecularly defined medium, SBX, associated with xenofree matrices, to maintain human embryonic stem cell (hESC) pluripotency as determined by phenotypic, functional and TLDA studies. This simple, inexpensive, and more physiological culture condition has been chosen because (1) it is xenofree and molecularly defined; it is devoid of albumin, which is a carrier of undefined molecules; (2) it maintains pluripotency, but very significantly reduces differentiation gene expression during hESC self-renewal, as compared to the widely used culture conditions tested so far; and (3) it can be further improved by replacing high concentrations of expensive additives by physiological concentrations of new factors. Xenofree molecularly defined media and matrices represent valuable tools for elucidating still unknown functions of numerous embryonic genes using more physiological culture conditions. These genes encode potential new factors controlling hESC self-renewal and pluripotency.

In a previous paper [8] we have shown that, with human CD 34+ cells grown in culture with serum, antisense TGF-β oligonucleotides significantly enhanced the frequency of colony formation by multilineage, 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-β is produced by a subpopulation of early progenitors. We now show that this effect is observed as well in serum-free culture which supports the development of mixed colonies. Therefore, the G0 phase control of some early progenitors, by an autocrine production of TGF-β, can be observed in vitro independently of serum or accessory cells.

Interleukin-3 (IL-3) is a hematopoietic growth factor suggested to be produced by activated T lymphocytes. Meanwhile, supernatants from human thymic stroma could promote the proliferation of myeloid stem cells. Thus, we investigated whether IL-3 accounts for this activity. Therefore, human thymic epithelial cells (TEC), fibroblasts, and adherent cells were isolated, and their culture supernatants assayed for myeloid colony promotion. Only supernatants from thymic epithelial cells supported colony-forming unit growth in semisolid media. This effect decreased following anti-IL-3 monoclonal antibody addition to these cultures. Furthermore, in situ hybridization showed the presence of IL-3 mRNA in epithelial cells. Effect of TEC culture conditions on IL-3 production by these cells was also studied. Together, these data show that IL-3 production is not the exclusive property of human activated T lymphocytes.

We have used antisense oligonucleotides to study the roles of transforming growth factor beta (TGF-beta) and the two antioncogenes, retinoblastoma susceptibility (Rb) and p53, in the negative regulation of proliferation of early hematopoietic cells in culture. The antisense TGF-beta 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-beta 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-beta. Anti-TGF-beta blocking antibodies, antisense TGF-beta, 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-beta negatively regulates the cycling status of early hematopoietic progenitors through interaction with the Rb gene product.

The mitogenic effect of human fibrinogen on the hemopoietic cell lines Raji and JM is mediated by a specific receptor with biochemical and functional properties different from those of the platelet fibrinogen receptor, the glycoprotein complex IIb-IIIa (Levesque, J.P., Hatzfeld, A., and Hatzfeld, J. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 6494-6498; Levesque, J.P., Hatzfeld, A., Hudry-Clergeon, G., Wilner, G.D., and Hatzfeld, J. (1987) J. Cell. Physiol. 232, 303-310). This work describes the identification of the mitogenic fibrinogen receptor (MFR) by two independent methods, affinity chromatography and covalent cross-linking. Affinity chromatography of surface-labeled cell extracts on fibrinogen-Sepharose revealed a 94 +/- 2-kDa membrane protein that bound specifically to fibrinogen-Sepharose only on cells that expressed the MFR. Its molecular mass was not modified after reduction. This was confirmed by cross-linking fibrinogen to surface-labeled Raji cells using the cleavable cross-linkers, ethyleneglycobis(succinimidyl succinate) and dithiobis(succinimidyl propionate). Complexes between fibrinogen and iodinated cell membrane proteins were immunoprecipitated by anti-fibrinogen antibodies. The biochemical cleavage of these immunoprecipitated conjugates gave rise to a 92 +/- 3-kDa membrane protein whose molecular mass was not modified after reduction. We conclude that fibrinogen binds specifically to a 92-94-kDa MFR which does not belong to the integrin family.

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-IFNalpha 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.

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-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 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 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: 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 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) 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 alpha6 and beta1 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-beta1, which is to promote undifferentiated keratinocyte amplification.

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.

Various protocols have been described to optimize gene transfer into hematopoietic cells. However, most of these methods do not specify whether they are associated with an improved transduction of the more primitive stem/progenitor cells, the best candidates for long-term engraftment. The majority of these primitive cells remains in quiescence because of the negative control of TGF-beta1, effective on these cells at low concentrations (10 pg/ml). In this study, CD34- cells were activated by a 10 h pretreatment with anti-TGF-beta1 followed by four successive retroviral supernatant incubations of 6 h each. After 12 h (two incubations), a significant increase in TGF-beta1 mRNA in CD34+ cells was observed. We wondered whether neo-synthesized autocrine TGF-beta1 could induce reversion to quiescence of the more primitive CD34+ cells transduced after one cell cycle. This would prevent their subsequent detection in a classic clonal assay. Using the HPP-Q assay comparing a rapid mixed colony assay with or without anti-TGF-beta1, we indeed observed, that in clonal growth conditions the more primitive transduced cells were activated and detectable only with anti-TGF-beta1. Therefore, this assay represents not only a rapid means to detect quiescent multipotent stem/progenitor cells but also a necessary step for the detection of the more primitive transduced cells which have returned to quiescence after retroviral induction of TGF-beta1 secretion.

In addition to its well-known functions in blood clotting and cell adhesion, fibrinogen has been reported to be a mitogen for lymphoid cell lines and for human hematopoietic progenitors. Two specific receptors, the mitogenic fibrinogen receptor (MFR) and intercellular adhesion molecule-1 (ICAM-1/CD54), have been identified as possible candidates for the mediation of the mitogenic effect of fibrinogen. However, it has been questioned whether the MFR and ICAM-1 are truly distinct molecules. Using an antiserum specific for the MFR, we demonstrate that the MFR is a cell surface molecule clearly distinct from ICAM-1. Both receptors can be expressed separately or coexpressed on different cell types. Moreover, they are regulated differently: ICAM-1 is calcium-dependent whereas the MFR is not and the MFR is down-regulated by fibrinogen whereas ICAM-1 is not. The inhibition by an anti-MFR serum of the mitogenic effect of fibrinogen confirms the mitogenic function of the MFR.

Using antisense TGF-s1 oligonucleotides (ODN) or anti-TGF-s serum in single cell and clonal culture of enriched progenitors from normal human bone marrow cells and umbilical cord blood, we have demonstrated that early hematopoietic progenitors produce autocrine TGF-s1. These cells could not be triggered into cycle by optimal concentrations of growth factors available, which included IL-3, IL-6, G-CSF, SL and Epo. Using medium with this cytokine cocktail supplemented with either antisense TGF-s 1 ODN or anti-TGF-s serum, we first estimated the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. The results showed that the capacity for long-term engraftment of a 100-200 ml sample of umbilical cord blood is greater than that of the volume of bone marrow typically used to engraft an adult. The release from quiescence of the early progenitor cells by anti-TGF-s serum was also used to improve gene transfer efficiency, since retroviral gene transfer requires a proliferative status of the target cell. High efficiency (95%) of gene transfer was obtained in early colony-forming unit-granulocyte-erythroidmegakaryocyte-macrophage (CFU-GEMM) with a stable gene expression. Development and size of the colonies were not affected by the retrovirally-mediated gene transfer. Among the normal hematopoietic progenitor populations, the earliest pluripotent cells are largely quiescent while a larger proportion of later, lineage-restricted cells are in active phases of the cell cycle (Lajtha and Schofield, 1974). Using antisense TGF-s1 oligonucleotides (ODN) or anti TGF-s serum in single cell and clonal culture of enriched progenitors from normal human bone marrow cells, we have demonstrated that early colony forming cells produce autocrine TGF-61. Indeed, antisense TGF-s ODN or anti TGF-s serum enhance colony formation from early progenitor cells such as CFU-GEMM and early BFU-E: we obtained twice more mixed colonies and erythroid bursts than in the control cultures.These colonies were larger (Hatzfeld et al., 1991). These cells could not be triggered into cycle by optimal concentrations of growth factors available, which included IL-3, IL-6, G-CSF and Epo. We have further shown that, although inclusion of SF increases the frequency of colony formation by early progenitor cells, it still does not reverse the effects of TGF-81 in culture and many cells remain quiescent (Li et al., 1994). We have therefore used cultures with our standard cytokine cocktail supplemented with SF and either antisense TGF-81 ODN or anti-TGF-s serum: 1) to compare the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. 2) to improve gene transfer efficiency since retroviral gene transfer requires a proliferative status of the target cell. To evaluate the proliferative capacity of bone marrow and umbilical cord blood progenitors, cells obtained from iliac crest of healthy individuals or from umbilical cord blood with the appropriate consent of the donors were purified as previously described, using the AIS procedure (SBA and CD34 CELLector flasks; Applied Immune Sciences Inc., Santa Clara, CA) (Cardoso et al., 1995). The isolated CD34+ population obtained by this procedure was 95 ± 3% pure. Cells were then cultured with the cocktail of cytokines in the presence of 21mers phosphorothioate TGF-s1 ODN or turkey anti-TGF-s blocking serum, either in liquid cultures for expansion or in clonogenic assays to evaluate the types of progenitors. The CD34+ CD38- population was purified by FACS. When cultured in the presence of IL-3, IL6, GM-CSF, SF, and Epo, colony formation by early progenitors (CFU-GEMM, HPP-CFC, and early BFU-E) in the cord blood CD34+CD38- fraction, but not in the CD34+CD38+ fraction, was significantly (1.5 to 2 fold) enhanced by antisense TGF-B1 ODN. Late progenitors (CFU-E, CFU-M, and CFU-G) in the same preparation were unaffected by the antisense ODN. These results suggest that some subpopulations of CD34+ CD38- cells are under the control of autocrine TGF-s. Cumulative evaluation of the respective number of colonies obtained after more than 35 days of culture leads to the conclusion that, although a typical bone marrow sample prepared for adult bone marrow transplant contains significantly more CD34+ cells than a typical cord blood sample (100-200 ml), the ability to generate early progenitors in primary culture are quite comparable for both samples. This results from the higher frequency of CD34+ CD38- cells in the cord blood preparations and from the higher proliferative potential of these cells in culture. Indeed, the CD34+ CD38- cord blood fraction yielded similar number of CFU-GEMM but almost 3 fold more early BFU-E and 2 fold more CFU-GM than did the corresponding bone marrow cell preparation (Cardoso et al, 1993). We took advantage of releasing the early progenitors from quiescence to improve transfer efficiency since the proliferative status of the target cell is a prerequisite for an efficient transfer. Purified cord blood CD34+ cells were prestimulated in the presence of cytokines and anti TGF-s serum. They were then transfected by cocultured for 40 h on 40% confluent NB16, a packaging cell line which express the E. Coli Bgalactosidase, kindly provided by Drs J.M. Heard and O. Danos (Ferry et al., 1991). Transduced CD34+ cells were then tested in clonogenic assays and colonies were stained with X-Gal as substrate (Strair et al., 1990). Results showed that addition of anti-TGF-s serum increased the transfer efficiency from 24% without anti-TGF-s serum up to 47.3% in the presence of anti-TGF-s serum. Interestingly, the effect of anti-TGF-s is most effective on the most immature progenitors, which develop into large mixed colonies (CFU-GEMM) with l-2xl05 cells. Anti-TGF-s serum pretreatment increases gene transfer in CFU-GEMM from 54 to 95%. It augments significantly the stability of gene expression in subpopulations of mixed colonies and the expression of the stably integrated recombinant provirus does not reduce their size. In conclusion, the elucidation of the mechanisms controlling cell proliferation, especially the role of inhibitors like TGF-s 1, will be ofinterest in clinical applications for transplantation with cell expansion as well as for gene therapy.

This European concerted action was established to promote collaborations and the exchange of information amongst European laboratories working on human haemopoietic stem cells. It is supported for three years by the European Commission, Directorate General XII under the direction of Dr Ch. Bardoux. There are, at present, over 56 contributing laboratories throughout Europe with 2 laboratories in Central and Eastern Europe joining through the PECO initiative in late 1994. The co-ordinator [Dr. S.M. Watt] is assisted in its management by Professor D. Weatherall and Dr. J. Hatzfeld and by an advisory board of 9 European Scientists whose names are listed in Table

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell 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 colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, 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 transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly 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 preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.

Ramalho-Santos et al . ([ 1 ][1]) and Ivanova et al. ([ 2 ][2]), comparing the same three “stem cells”— embryonic stem cells (ESCs); neural stem cells (NSCs), referred to as neural progenitor/stem cells (NPCs) in the present study; and hematopoietic stem cells (HSCs)—with their

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.

In comparing spleen cells of inbred and congenic mice for spontaneous capacity to lyse cells of the BALB/c leukemia RLmale1 in vitro, we found that the activity of 129 spleen cells was more than double that of 129-Gix- spleen cells. The only known difference between these two strains is that 129-Gix- mice express no known demonstrable gp70 or p30, whereas 129 mice express both these MuLV-related components as mendelian traits not associated with the production of virions. We infer that MuLV-related components at the cell surface are concerned in effector-target interactions leading to cytolysis under the conditions described. Although the congenic strains B6 (Gix-) and B6-Gix+ differ likewise in expression of the type-variant Gix-gp70, both strains express a second type-variant of gp70. The lytic activity of spleen cells of these two strains for RLmale1 cells was equally high, suggesting that involvement in lytic effector-target interactions is common to gp70 molecules in general. When used as targets rather than as effectors 129 spleen cells were more sensitive to lysis than 129-Gix- spleen cells. Pre-exposure to gp70, purified from R-MuLV, rendered splenic effector cells less lytic. Pre-exposure to gp70 also rendered RLmale1 target cells less sensitive to lysis. One explanation of these findings is that both target cells and effector cells express gp70 and also receptors for gp70 and that this is the basis of mutual cellular recognition leading to lysis in the circumstances described.

Purified fibrinogen at concentrations of 3-30 nM has been found to stimulate continuous growth of human lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphoma-derived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 10(5) cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, approximately equal to 50 granulocyte-macrophage colonies per 10(5) cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.

Using the indirect immunoperoxidase technique with optical and electron microscopy, we have localized both fetal aldolases A and C from regenerating rat liver in Kupffer and endothelial cells (by contrast with the localization of aldolase B in hepatocytes only). These results have been confirmed by biochemical methods. The localization of the 3 aldolases differs widely from that observed in fast-growing hepatoma and suggests that control mechanisms of gene regulation are different in cancer and in liver regeneration.

A gradient has been developed for separating free ligand from ligand bound to cells growing in suspension. This method can be used with all kinds of ligand but it is particularly useful for those ligands having the tiresome tendency to adhere to the cells non-specifically or to polymerize by themselves. This is the case of fibronectin, fibrinogen, immunoglobulins and many other plasma proteins. The gradient consists of two layers: an upper aqueous phase and a lower hydrophobic organic phase. The aqueous phase, a sucrose buffer, allows the cells to become well dispersed before they enter the hydrophobic phase which excludes the free ligand efficiently. This reduces non-specific binding and allows the accurate measurement of specific binding which could not be obtained with a gradient made of a single phase. Depending upon the size and the density of the cells, and the nature of the ligand, the assay method can be modified by changing the density or the nature of the hydrophilic and hydrophobic phases.

We compared the in vitro allogeneic response of human lymphocytes cultured in hormonally defined medium (HDM) with that of those cultured in plasma-containing medium (PCM). Differences occurred only in secondary MLR or PLT and in cell-mediated cytotoxicity in terms of specificity, which was either greater or modified when HDM was used.

Fibrinogen is absent in serum but is a major component of plasma, where its concentration is about 3 mg/ml. Blood clotting results from the polymerisation of fibrinogen into fibrin (l). Here we present evidence that fibrinogen may play another role by stimulating the proliferation of certain human hemopoietic cells. A strong proliferative response of certain cell lines resulted from the synergism elicited by the addition of fibrinogen (3–30 nM) to transferrin in defined medium. Fibrinogen was also found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semi-solid media containing serum. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin thus eliminating fibrin as a source of the mitogenic effect.

The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg-Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation.

Spleen cells from an SJL mouse immunized with B10.S spleen cells were fused with the nonsecretor myeloma line NS.1. One established hybrid cell line continuously secreted antibody that recognized a new antigenic specificity, tentatively called "Ly-m11." This newly found antigen is detectable on nearly 100 percent of spleen and lymph-node cells, 70 percent of bone-marrow cells, and 20 percent of thymus cells by direct cytotoxicity assays, and on the cells derived from kidney and liver. Strains that are Ly-m11 (+) include C57BL/6, C57BL/10J, B10.S, C57BR/cdJ, C57L/J, and C57BL/KsJ. Other mouse strains so far tested are Ly-m11 (-). The strain distribution pattern distinguished Ly-m11 from any known murine lymphocyte alloantigens, but it follows the H-3 alpha haplotype which is defined by skin transplantation. Linkage tests of nine congenic strains of H-3 and/or H-13/alpha loci and five recombinant inbred lines including CXB, BXH, AKXL, SWXL, and BXD revealed no recombinations between H-3 and Ly-m11 loci on chromosome 2. This newly discovered Ly-m11 alloantigen could itself constitute a minor histocompatibility antigen detectable by serological means.

Summary The presence of aldolases A and B in the mammalian fetal liver has been well established. In this work, we give evidence in favor of the presence in the rat fetal liver of the third known type of aldolase, aldolase C, hybridized with aldolase A. This evidence comes from : u — electrophoresis : similar anodic isozymic migration of the enzymes are observed in both brain and fetal liver. — immunology : Anti C antiserum inhibited the fetal liver aldolase activity. The evolution of the three types A, B and C of aldolases from the 15th day of pregnancy to the third day after birth is characterized by a decrease of type A and C whereas type B increases up to about the same level as in the adult. The same methods show that during the same time interval, fetal aldolase in rat brain has reached the same level as in the adult with only small variations. Therefore, aldolase C in the mammalian as in the birds is present not only in the brain but also in several embryonic tissues.

The presence of aldolase C in rat fetal liver is a controversial problem with seems to stem from the methods used to study it as well as the cellular composition of the liver. It is discussed in the light of new data obtained from enzymatic, electrophoretic and immunological methods.

INTRODUCTION Transforming Growth Factor-beta1 is known to maintain primitive human hematopoietic stem/progenitor cells in a quiescent state. However, its specific role in the control of distinct progenitor cell types needs to be further elucidated. In this study, we have investigated the dose-response effect of TGF-beta1 on progenitors ranging from primitive high proliferative potential (HPP)-Mix, -GM or -BFU-E to later BFU-E, CFU-G or CFU-M. MATERIALS AND METHODS A clonal semi-solid assay has been used to analyze the effects of a TGF-beta1 blocking antibody (anti-TGF-beta1) or that of active TGF-beta1 added to the medium at concentrations from 10-3000 pg/ml, on these different hematopoietic stem/progenitor cell types. RESULTS AND CONCLUSION A preferential growth inhibitory effect on the earlier progenitors was observed when low concentrations of TGF-beta1 (10-300 pg/ml) were used. Concentrations of 10-30 pg/ml TGF-beta1 were sufficient to inhibit 90% of the primitive multipotent HPP-Mix, while 100-300 pg/ml TGF-beta1 were required to inhibit 70% of the bipotent HPP-GM and early HPP-BFU-E. TGF-beta1 did not significantly inhibit or activate the growth of later CFU-G and CFU-M, even when added at concentrations 10-100 fold higher. In contrast, a significant growth-inducing effect of very low TGF-beta1 concentrations (< or =30 pg/ml) on a subset of later BFU-E was observed and cannot be explained by a switch of early into later BFU-E. These results emphasize the polyfunctional role of TGF-beta1 in the regulation of hematopoiesis and the need for low, physiological concentrations of TGF-beta1, when studying both the stem cell compartment and more mature progenitor cell subpopulations.

Leupeptin, a thiol- and serine-proteinase inhibitor of low molecular weight, quickly enters viable cells. This property has been used to protect cells during thawing against intracellular proteolytic activities released by injured lysosomes. The bone marrow nucleated cells were frozen without rate-controlled freezing devices. Concentrations ranging from 0.1 to 1 microM of leupeptin allow to recover 87% of the most immature multipotent bone marrow progenitors which can develop in vitro into large multilineage colonies, instead of 58% recovery without leupeptin. The protective effect of leupeptin is particularly useful to freeze cells difficult to cryopreserve or when freezing-control equipment are not available.

An isolation procedure for rat brain aldolase C has been developed which also permits the isolation of aldolase C from experimental hepatomas. Certain enzymatic properties (specific activity and Michaelis constant towards the two specific substrates: fructose 1,6-bisphosphate and fructose 1-phosphate) and physico-chemical properties (molecular weight, N-terminal amino-acid) of the two enzymes have been studied and compared. Moreover, an amino-acid analysis has been carried out for rat brain aldolase C. Within experimental errors, the two enzymes appear to be identical.

Hematopoiesis is a remarkable cell-renewal process that leads to the continuous generation of large numbers of multiple mature cell types, starting from a relatively small stem cell compartment. A highly complex but efficient regulatory network is necessary to tightly control this production and to maintain the hematopoietic tissue in homeostasis. During the last 3 decades, constantly growing numbers of molecules involved in this regulation have been identified. They include soluble cytokines and growth factors, cell–cell interaction molecules, and extracellular matrix components, which provide a multifunctional scaffolding specific for each tissue. The cloning of numerous growth factors and their mass production have led to their possible use for both fundamental research and clinical application.