Thérapie cellulaire | EPFL Graph Search

vignette|Thérapie par lymphocytes T adoptifs. Dans le cadre des « biothérapies », la thérapie cellulaire vise à soigner un organe ou un organisme par l'apport de cellules, obtenues la plupart du temps à partir de cellules souches, pour remplacer ou suppléer des cellules défaillantes. Le nombre de pathologies ciblées par cette biothérapie est important : Alzheimer, Parkinson, diabète, leucémie Elle pourrait également faciliter les greffes ou la restauration d'organes. Il faut pour cela réveiller les fonctions de codage de la cellule, les orienter vers le but recherché, et que les cellules ou organes produits soient acceptés dans l'organisme receveur (qui peut aussi être donneur). Ces cellules souches peuvent être la base de travaux de thérapie génique, on parle alors de thérapie génique ex vivo. Dans le cadre de traitements non conventionnels, il existe des ou reposant sur l'injection de protéines animales pour de supposées bénéfices anti-vieillissement voire anti-cancer. Aucune étud

Optimization of human erythropoietin secretion from MLV-infected human primary fibroblasts used for encapsulated cell therapy

Patrick Aebischer

BACKGROUND: The transplantation of encapsulated cells genetically engineered to secrete human erythropoietin (hEpo) represents an alternative to repeated injections of the recombinant hormone for the treatment of Epo-responsive anemia. In the present study, the ability of primary human foreskin fibroblasts to secrete high levels of hEpo and the importance of cis-acting elements and infection conditions on transgene expression level were assessed. METHODS: The transduction efficiency was first evaluated with beta-galactosidase (LacZ)-encoding retroviral vectors derived from the murine leukemia retrovirus (MLV) pseudotyped either with an amphotropic envelope or with the G glycoprotein of vesicular stomatitis virus (VSV-G). Human fibroblasts were then infected with an amphotropic hEpo-expressing retroviral vector, which was modified by insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE) and a Kozak consensus sequence (KZ). Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells. The survival and the transgene expression of these fibroblasts were finally evaluated in vivo. The cells were encapsulated into microporous hollow fibers and subcutaneously implanted in nude mice. RESULTS: A secretion level of approximately 5 IU hEpo/10(6) cells/day was obtained with the basal vector. A 7.5-fold increase in transgene expression was observed with the insertion of WPRE and KZ elements. Finally, according to the optimization of infection conditions, we obtained a 40-fold increase in hEpo secretion, reaching approximately 200 IU hEpo/10(6) cells/day. The in vivo experiments showed an increase in the hematocrit during the first 2 weeks and elevated levels exceeding 60% were maintained over a 6-week period. CONCLUSIONS: These results indicate that primary human fibroblasts represent a promising source for encapsulated cell therapy.

2003

Isolation and characterisation of a porcine cardiac adherent cell population from heart biopsies

Christelle Hasselmann

Cardiac diseases represent the major cause of mortality in the Western world. The discovery of the promising human Cardiac Adherent Proliferating (CAP) cells for a potential cardiac cell therapy by the Tissue Engineering group induced the need of a reliable animal model. The aim of the project is to identify a porcine cardiac cell population having similar characteristics as human CAP cells. Here a protocol for isolating cells from porcine heart biopsies have been established, as well as the in vitro expansion method. Additionally, the characterization of those expanded porcine cardiac cells was accomplished by growth kinetics study, FACS analysis, multilineage potential and myogenic induction assays. In this study we identified a not yet described porcine cardiac adherent cell population directly isolated from biopsies within the inner right ventricle. With almost

5\times {10}^{7}

expanded cells after 30 days in culture, porcine cardiac cells present a high rate of proliferation in vitro. Porcine cardiac cells maintain a swirl-like pattern until 5-6 days in culture, and become fibroblast-like with a parallel organization when confluent. FACS analysis indicated that porcine cells are mainly CD90+ in opposite to human CAP cells. Both species show a similar CD45- and CD44+ profile. The multilineage assay showed no histological differentiation into fat, bone and cartilage on porcine cardiac cells like on human CAP cells. However gene expression analysis on porcine cardiac cells indicated that aP2 was upregulated in population undergoing an adipogenic induction, as well as osteopontine expression in osteogenic induction population. Porcine cardiac cell myogenic induction potential was tested on C2C12 myogenic mouse cells. The mouse cells differentiated in muscle in vitro thanks to the influence of those cardiac cells. Both cell populations have similar characteristics and the main distinction is the different CD90 expression profile. Those results encourage to realise additional studies, to confirm that those isolated cells could be a reliable model of human CAP cells.

2009

Bioengineering human bone marrow models to elucidate the triggers of the stem cell niche

Queralt Vallmajó Martín

The bone marrow (BM) microenvironment, or niche, significantly affects behaviors of its resident stem cell populations. Disruptions in the BM niche contribute to a number of severe clinical pathologies. Discovery of novel therapeutic strategies for BM-related diseases necessitates development of superior preclinical models to study parameters affecting BM homeostasis. To address this, a poly(ethylene glycol) hydrogel featuring a particular crosslinking chemistry based on the enzyme transglutaminase factor XIII, dubbed TG-PEG, was utilized to engineer humanized bone marrow models. Its modular nature was exploited throughout this work to systematically evaluate contributions of biophysical, cellular, and biochemical BM niche components to stem cell biology. Stiffness of TG-PEG hydrogels was optimized for each cell type and individual application explored. For bone formation by bone marrow stromal cells (BMSCs), an intermediate stiffness was optimal and significantly improved total bone volume over softer TG-PEG gels or natural materials. Results indicated that stability of the scaffold was more important than their chemical and biological properties. Alternatively, for hematopoietic stem and progenitor cells (HSPCs), softer gels were optimal for preserving multipotency while promoting cell proliferation. A number of BM-specific stem and progenitor cell types with richly diverse functions were explored. Skeletal stem cells (SSCs) encapsulated in TG-PEG hydrogels demonstrated their ability to form de novo bone in healing critically sized calvaria defects in mice. Next, subcutaneous implantation of BMSC-laden gels revealed their significant contribution to formation of ossicles. For some BMSC donors, ossicles could be formed in absence of bone morphogenetic protein (BMP)-2 offering a novel tool to evaluate their intrinsic capacity. Later, titration of BMSC seeding densities and BMP-2 doses yielded optimal conditions for robust BM formation, regardless of donor, enabling reproducibility for follow-on studies involving BM models. In humanized mice, homing of HSPCs to BMSC-laden TG-PEG gels was observed. Finally, humanized xenograft models were employed to determine vulnerability of the BM niche to subversion by cancer cells simulating osteotropism for both leukemia and breast cancer metastasis. Molecular factors known to trigger particular cell reactions within the niche were also investigated. Including RGD cell-adhesion ligands, as well as matrix metalloproteinase (MMP)-susceptible crosslinks to render the gels degradable, were critical for facilitating migration of recruited BM cells from murine hosts in xenograft studies. Hyaluronic acid (HA), when added to the hydrogel backbone, conferred superior engraftment to transplanted BMSCs and afforded reduced immunogenicity. Incorporation of the Notch-signaling ligands Jagged1 or DLL-4 into soft TG-PEG gels during in vitro HSPC expansion substantially stimulated proliferation, but recovered cells could no longer ensure long-term reconstitution. Aging, trauma, or cancer can cause disruptions of the tightly controlled balance between cells and their microenvironment in the BM niche. This body of work presents a number of bioengineered tools optimized for studying how niche components are affected by or contribute to such pathologies. Further work with these or similar models will no doubt highlight key clinically relevant targets and inform novel therapeutic approaches.

EPFL2019