Ma-n Li

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).

Optimization of Physiological Xenofree Molecularly Defined Media and Matrices to Maintain Human Embryonic Stem Cell Pluripotency

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.

Inhibition of autocrine TGF-ß in CD34+ human progenitor cells reveals their potentiality to engraft adults and improves gene transfer efficiency

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.