Véronique Decot

Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Whartons Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organs reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

Natural-killer cell amplification for adoptive leukemia relapse immunotherapy: Comparison of three cytokines, IL-2, IL-15, or IL-7 and impact on NKG2D, KIR2DL1, and KIR2DL2 expression

OBJECTIVE Natural killer (NK) cells are a lymphocyte subset that, in a hematopoietic stem cell transplantation setting, mediates a graft-vs-leukemia effect without any graft-vs-host disease. We aimed to evaluate an isolation method that can be used with Good Manufacturing Practices-grade reagents and to compare three cytokines for expansion in order to design future clinical protocols based on donor NK-cell infusions to cure relapse after allograft. MATERIALS AND METHODS NK cells were enriched using a CD3/CD19 depletion method and expanded for 13 days in the presence of 2, 10, and 50 ng/mL interleukin (IL)-2, IL-15, or IL-7. NK-cell cytotoxicity was evaluated after isolation and culture. Expression of NKG2D, KIR2DL2, and KIR2DL1 was monitored during expansion. RESULTS Highly T- and B-cell-depleted NK cells were obtained and enriched 2.6-fold. The optimal cytokine concentration for expansion was 10 ng/mL for IL-2 or 50 ng/mL for IL-15. NK-cell cytotoxicity was significantly improved after an overnight incubation with 10 or 50 ng/mL IL-2 or with 2, 10, or 50 ng/mL IL-15, and after 13 days with 50 ng/mL IL-15. The use of a combination of IL-2 and IL-15 showed no additional benefit and negative results were obtained with IL-7. The three NK cell receptors were significantly upregulated after culture, mainly with IL-2 or IL-15. CONCLUSION In our study, 10 ng/mL IL-2 or 50 ng/mL IL-15 were the optimal concentrations for expansion and were equivalent in significantly enhancing cytotoxicity and modifying NK-cell receptor expression patterns.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-γ (IFN-γ) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01±0.84×106 total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56%±20.8%, yield=51%±32.4%) but also CD8+ (mean=42%±27%, yield=56%±39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-γ and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation.

BACKGROUND Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. METHODS EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. RESULTS Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p=0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis, four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10(6)PBMC. CONCLUSION Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

Research on stem cells as candidates to be differentiated into hepatocytes

Liver diseases have become one of the most important causes of morbidity and mortality in the world. Cell therapy and liver transplantation are though to be two treatment options well accepted. However, the shortage of cells sources in cytotherapy and the lack of liver donor in liver transplantation are the major obstacles for the performance of these treatment methods. It urged us to find new origins of extra-hepatic cells. A number of recent studies show that extra-hepatic mesenchymal stem cells (MSC) from different tissues can be differentiated into hepatocytes like cells (HLC). Several hepatic differentiation protocols of MSC have been published in recent years, based on cellular stimulation with exogenous cytokines/growth factors, co-culture with fetal or adult hepatocytes, 2- or 3-dimensional (2D, 3D) matrices to favor differentiation. Independently from the starting stem cells population used, some minimal criteria must be fulfilled to ensure therapeutic success: in vitro expandability, expression of hepatic like surface markers, with hepatic cell functions, and minimal or absent immunogenicity in the recipient host. In this review, we focused on stem cells originated from bone marrow, umbilical cord and adipose tissue which are widely investigated in recent years and have been proved to have liver regenerative potential, the factors used to differentiate stem cells to hepatocyte-like cells and the methods used to investigate these cells.

In vitro initial expansion of mesenchymal stem cells is influenced by the culture parameters used in the isolation process

In the last years, there were many studies based on the use of human bone marrow mesenchymal stem cells (hMSCs) in cell therapy and tissue engineering. Although hMSCs can be easily obtained and expanded in culture, a large number of cells are often needed. The expansion of hMSCs depends on the culture conditions, such as media, cell density or culture flasks. Moreover, growth factors are often added to improve cell proliferation. In this study, we compared the effect of two culture media (DMEM and alpha-MEM), two culture flasks (75 or 25 cm2) and two different mononuclear cell seeding densities (1 x 10(4) or 5 x 10(4) MNC/cm2) on the isolation of hMSCs from bone marrow samples and analyzed if the isolation conditions affected the expansion of these cells in the first two passages. Experiments were performed without the addition of exogenous growth factors. Our results showed that alpha-MEM is the optimal culture medium for both, isolation and expansion of mesenchymal stem cells. Moreover, the cell seeding density of 50,000 MNC/cm2 in 25 cm2 culture flasks seems to be the best condition for the isolation step.

Introduction to tissue engineering and application for cartilage engineering

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.

Regulatory aspects of cellular therapy product in Europe: JACIE accreditation in a processing facility.

In 1997, the Joint Accreditation Committee ISCT & EBMT (JACIE) was created. The following year, it approved the first edition of standards for haemopoietic progenitor cell collection, processing and transplantation. The purpose of the standards is to ensure a minimal level of quality, alertness and homogeneity in the implementation of autologous and allogeneic haemopoietic stem cell transplantation (HSCT) programme in onco-hematology. The acquisition of accreditation is based upon the system of examination by trained medical professionals according to countries endorsements with the national regulation obligations applicable to HSCT. In 2008, the fourth edition has been launched. The range of application of the standards comprises both donors and recipients, and all phases of collection, processing, storage and administration of haemopoietic progenitor cells. Among the accredited processing facilities, a few have been integrated JACIE standards into their existing management quality system which is inspected by national health authority. In France, the comparison between JACIE standards and the good manufacturing practices of cellular therapy product reveals some common points and some differences to apply.

Human Stem Cells and Articular Cartilage Tissue Engineering

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on cartilage tissue engineering combining cells, scaffold materials and environmental factors. This paper focuses on the main parameters in tissue engineering and in particular, on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. We discussed the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarized the advantages and disadvantages of these cells in articular cartilage engineering.