Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium.

Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(®)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(®)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(®)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(®)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(®)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.

c-fos mRNA constitutive expression by mature human megakaryocytes.

By in situ hybridization with a c-fos probe, we have shown that human bone marrow megakaryocytes cultured in the presence of 20% aplastic anemia plasma constitutively express c-fos mRNA. At day 0, megakaryocytes are mostly immature and only 3% of them are labeled. The number of labeled cells reached 23% after 12 days of culture. Interleukin 3 (IL-3) and IL-6 added together at day 10 further increased this number to 31% 2 days later. Mature labeled megakaryocytes were more numerous and more strongly labeled than immature ones. These results suggest that c-fos could play a role in megakaryocytic terminal differentiation, either in the polyploidization or in the thrombopoietic function unique to these cells.

Increased stable retroviral gene transfer in early hematopoietic progenitors released from quiescence.

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.

Fibrinogen cooperates with cytokines to induce interleukin-6 receptor mRNA expression in human hematopoietic CD34+ progenitors.

We have previously demonstrated that purified human fibrinogen (Fg), a major plasma component removed during serum preparation, shows mitogenic properties towards lymphoma cells and normal human hematopoietic progenitors. Indeed, adding Fg with IL-3 to a serum-containing medium stimulates growth of human CD34+ progenitors. In this report, we show in serum-free medium, that this stimulating effect only occurs in the presence of IL-6. To clarify the cooperative effect between Fg and IL-6, the kinetics of IL-6 receptor (IL-6R) mRNA expression in CD34+ cells have been analyzed by semi-quantitative in situ hybridization. In the presence of both IL-3 and Fg, more cells express IL-6R mRNA, and this expression per cell is significantly greater than with each factor added separately. These results suggest that Fg does not promote the growth of normal cells by itself, but sensitizes the cells to cytokines. Fg behaves not as a "progression" factor but as a typical "competence" factor, which induces a faster and greater IL-6R expression in early human hematopoietic progenitors by cooperating with other cytokines.

[Quality control of defrosted cord blood units: results from an inter-laboratory study]. / Contrôle de qualité des greffons de sang placentaire décongelés : résultats d'une étude multicentrique.

PURPOSE: Today, haematopoietic stem cell graft from placental blood concerns more than 15 % of allogeneic grafts. An inter-laboratory study of the quality control of defrosted cord blood units has been coordinated by the French society for cell and tissue bioengineering (SFBCT), with the cord blood bank of Bourgogne Franche-Comté and controlled by the French health products safety agency (Afssaps). The aim of this study is to ensure the inter-laboratory reproducibility of the quality controls practised by the banks during defrosting. The cellular outputs were analyzed according to the defrosting techniques, according to the method used in flow cytometry: single-platform (SP) versus double-platform (DP), or the product nature, i.e. in total blood or miniaturized. METHODS: Forty-two units of placental blood (USP), which were out of range were provided for defrosting to 14 participating sites. USP were defrosted and controlled according to the procedures of each bank. Once the USP is defrosted, a part of the product was controlled by the site and the other part by Afssaps. Following controls were carried out: numeration of the total nucleated cells (TNC) and of CD34+ cells (made by a SP method in Afssaps) and functional assay. RESULTS: Concerning TNC, the defrosting sites obtained a cellular output of 94 %+/-28 in day 0 compared with an output of 72 %+/-24 in Afssaps showing a rather good stability of the USP transmitted with an average deviation of 23 %+/-22. The freezing process with or without reduction of volume does not affect this variation. Concerning the numeration of CD34+ cells, the average deviation between the participating sites and Afssaps was 29 %+/-23 compared with 21 %+/-16 for the sites using a SP method against 47 %+/-25 for those using a DP method. The CD34+ outputs are equal to 82 % +/- 60 in day 0 for the participating sites against 52 %+/-20 for Afssaps. For the sites using a DP method, it is stressed that this output is particularly high with a rate of 126 %+/-90 (n=15) whereas it is 62 %+/-20 (n=32) for the sites using a SP method. CONCLUSION: These results underline a good stability of viable CD34+ cells and a greater reliability of the SP methods for the CD34+ cell numeration for these defrosted USP. Lastly, the results of the functional assay regarding the average clonogenicities (equal to 15 %) reinforce the conclusions on the quality of the defrosted products.

Cryopreservative effect of leupeptin on early human bone marrow progenitors.

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.

p21(cip1) mRNA is controlled by endogenous transforming growth factor-beta1 in quiescent human hematopoietic stem/progenitor cells.

Transforming growth factor-beta1 (TGF-beta1) 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-beta1 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-beta1 has downmodulated the cytokine receptors. These cells can be released from quiescence by neutralization of autocrine or endogenous TGF-beta1 with a TGF-beta1 blocking antibody or a TGF-beta1 antisense oligonucleotide. In nonhematopoietic systems, TGF-beta1 cooperates with the cyclin-dependent kinase inhibitor, p21(cip1), to induce cell cycle arrest. We therefore analyzed whether endogenous TGF-beta1 controls the expression of the p21(cip1) in the CD34(+) undifferentiated cells using a sensitive in situ hybridization method. We observed that addition of anti-TGF-beta1 is followed by a rapid decrease in the level of p21(cip1) mRNA whereas TGF-beta1 enhances p21(cip1) mRNA expression concurrently with an inhibitory effect on progenitor cell proliferation. These results suggest the involvement of p21(cip1) 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 p21(cip1) observed in response to TGF-beta1 allows us to further precise the working model of high proliferative potential-quiescent cells.

CSF-1 control of C-FMS expression in normal human bone marrow progenitors.

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.

IL-3, GM-CSF and CSF-1 modulate c-fms mRNA more rapidly in human early monocytic progenitors than in mature or transformed monocytic cells.

We have previously shown that a low concentration of CSF-1 (1 U/ml) can trigger human immature monocytic progenitor proliferation in the presence of low concentrations of IL3 (1.7 U/ml). No c-fms down-regulation was observed during this early cell activation. In contrast, 20 U/ml of CSF-1, active on late monocytic cell growth, down-regulated c-fms mRNA expression in immature progenitors and monocytes derived from bone marrow CD34+ cells in culture. We have now extended this study to include the effects of various concentrations of GM-CSF, IL3 and G-CSF on c-fms expression. We observed that high doses of GM-CSF or IL3 down-modulated c-fms mRNA, whereas low doses of GM-CSF or IL3, which were active on early monocytic growth, had no such effect. Similar results were observed at the protein level. In contrast, whatever the concentration, G-CSF had no effect on c-fms mRNA or protein levels. We further observed that the more immature the c-fms expressing progenitors, the faster the down-modulation of this receptor. This was observed within less than 1 hour for immature bone marrow cells, 6 hours for peripheral blood monocytes and even longer for transformed monocytic cells. These results suggest that oncogene expression can be regulated much more rapidly in immature progenitors than was previously observed in mature cells or transformed cell lines.

High proliferative potential-quiescent cells: a working model to study primitive quiescent hematopoietic cells.

Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-beta1 (TGF-beta1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-beta1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-beta1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-beta1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-beta1 but rapidly up-modulated by anti-TGF-beta1. (3) Anti-TGF-beta1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3(low) and IL6-Rlow). According to these results, we propose the working model of 'High Proliferative Potential-Quiescent cells' to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-beta1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.

Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

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.

Purification and release from quiescence of umbilical cord blood early progenitors reveal their potential to engraft adults.

Steel factor (SF) increases the frequency of colony formation by CD34+ CD38- cycling cells, but it does not reverse the effect of an autocrine production of transforming growth factor (TGF)-beta 1 by early progenitors of the stem cell compartment. We have used optimal culture conditions supplemented with SF and anti-TGF-beta serum to estimate the proliferative capacity and ability to generate early progenitors in long-term cultures of bone marrow and umbilical cord blood cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU), twice as many granulocyte-macrophage (GM) CFU, and three times as many erythroid burst-forming units as the same population from an average bone marrow sample used in adult transplantation. These results suggest that umbilical cord blood is a suitable source of cells for adult transplantation.

[Positive and negative controls on hematopoietic progenitor development: models and facts]. / Contrôles positifs et négatifs du développement des progéniteurs hématopoïétiques: modèles et faits.

The stochastic model of stem cell differentiation is in accord with experimental findings but does not explain hematopoietic homeostasis. We discuss how positive and negative controls by cytokines and inhibitors could maintain homeostasis, even though progenitor commitment towards the various hematopoietic lineages may be stochastic.

Early CD34high cells can be separated into KIThigh cells in which transforming growth factor-beta (TGF-beta) downmodulates c-kit and KITlow cells in which anti-TGF-beta upmodulates c-kit.

We have previously shown that early human CD34high hematopoietic progenitors are maintained quiescent in part through autocrine transforming growth factor-beta 1 (TGF-beta 1). We also demonstrated that, in the presence of interleukin-3, interleukin-6, granulocyte colony-stimulating factor, and erythropoietin, TGF-beta 1 antisense oligonucleotides or anti-TGF-beta serum have an additive effect with KIT ligand (Steel factor [SF]), which suggests that they control different pathways of regulation in these conditions. This finding also suggests that autocrine TGF-beta 1 might suppress c-kit expression in primitive human hematopoietic progenitors. We have now distinguished two subpopulations of CD34high cells. One subpopulation expresses a c-kit mRNA that can be downmodulated by exogenous TGF-beta 1 within 6 hours. Another subpopulation of early CD34high cells expresses a low or undetectable level of c-kit mRNA, but its expression can be upmodulated within 6 hours by anti-TGF-beta. These effects disappear 48 hours after induction and cannot be maintained longer than 72 hours, even if TGF-beta 1 or anti-TGF-beta serum are added every day. Similar kinetics, although delayed, are observed with KIT protein expression. On the contrary, no specific effect of TGF-beta 1 was observed on c-fms, GAPDH, and transferrin receptor gene expression in these early progenitors. These results clarify the complex interaction between TGF-beta 1 and SF in normal early hematopoietic progenitors. SF does not switch off the TGF-beta 1 inhibitory pathway. Autocrine TGF-beta 1 appears to maintain these cells in a quiescent state, suppressing cell division by downmodulating the receptor of SF, a key cytokine costimulator of early progenitors.