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New Advanced TherapY medicinal Product for Cartilage defect treatment with extemporaneous association of a scAffold and Wharton’s jelly mesenchymaL stromal cells – ATYPiCAL


New Advanced TherapY medicinal Product for Cartilage defect treatment with extemporaneous association of a scAffold and Wharton’s jelly mesenchymaL stromal cells.

Clinical grade production of WJ-MSCs, chondrogenic differentiation of WJ-MSCs in a stratified scaffold and behaviour of immune properties of WJ-MSCs during differentiation

1.The first step consisted in the production of WJ-MSC in clinical grade conditions. 8 productions among the 12 in the ANR project are already cryopreserved.<br />2. The second step consisted in the construction of the stratified scaffold, functionalized or not with BMP, associated to WJ-MSC (WJ-MSC/Alg-HA, WJ-MSC/Alg-HA/Nano-M1 +/- BMP2) and the investigation of the construction impact onto chondrogenic differentiation. We also aim at studying the becoming of the immunomodulatory properties of WJ-MSC embedded into our 3D scaffolds. This was recently performed for the first production of WJ-MSC with the scaffolds : Alg/HA and Alg/HA/NanoM1 without BMP2. Samples were collected to perform all the analysis and only preliminary results are availalble.<br />3. The third step will check, in vitro, that immune response currently observed in an allogeneic context does not negatively interfere with chondrogenic differentiation. The allogeneic context was first mimicced by addition of IFNg and TNFa during 48 hours. preliminary results are available.<br />4. The last step will consist in an in vivo study where extemporaneous WJ-MSC/Alg-HA/Nano-M1 +/- BMP2 association will be implanted into relevant animal models to study the biocompatibility, the chondrogenic differentiation and immunological properties. This step was not initiated.

Clinical grade generation of WJ-MSC requests GMP facilities and culture media and materials available for clinical use.
Flow cytometry with and without intra-cellular staining was performed, samples were collected for Elisa dosages and Mixed Lymphocyte reactions were performed in an unusual context, as it was done using inserts with Nano-M1 membrane.
For chondrogenic differentiation, flow cytometry was performed and samples for histology and molecular analysis were prepared and preserved in order to be further analyzed.
Nano-M1 membrane were provided by ARTiOS Nanomed; the production process is protected by a patent.

The main results generated by this ANR project at this time are :
- the definition of criteria for selection of umbilical cords, allowing a higher proliferation rate and an increased chondogenic differentiation ability. This work was recently published (see below).
- The generation of WJ-MSCs in clinical grade conditions : 8 umbilical cords were processed until today and WJ-MSCs were generated and cryopreserved. The process proposed by Capelli et al was optimized.
- The use of clinical grade reagents induced some testings especially for Alginate which viscosity is critical. Moreover, as the gelification process was different that the one performed at research scale, some validations were performed.
- Encouraging results were observed when the first chondogenic differentiation was performed : (i) chondrogenic differentiation was for the moment controlled with a high expression in flow cytometry of Collagen II, (ii) the low or absence of expression of co-stimulatory molecules and the detection of IDO, HLA-G and PDL-1 even at the end of the chondrogenic differentiation (iii), the non immunogenicity and the immunomodulatory properties until d14 of the chondrogenic differentiation. The preliminary results obtained at d30 are not in accordance with those results but technical difficulties impaired the test.

The future prospects will be :
- to finalyze this first manipulation that was very useful to test the coordination between the different partners.
- to perform the next manipulations using the other WJ-MSC batches that are already cryopreserved, in order to confirm these very preliminary results.
- to complete our manipulations with the conditions and the tests that were not yet performed: for example using the BMP2 functionalized Nano-M1 membrane, studying the T reg subpopulation, etc...
- to prepare the in vivo expriments

Umbilical cord-derived mesenchymal stromal cells: predictive obstetric factors for cell proliferation and chondrogenic differentiation.
Avercenc-Léger L, Guerci P, Virion JM, Cauchois G, Hupont S, Rahouadj R, Magdalou J, Stoltz JF, Bensoussan D, Huselstein C, Reppel L.
Stem Cell Res Ther. 2017 Jul 5;8(1):161.

The development of tissue engineering (TE) now allows considering the clinical use for osteoarticular defects of Mesenchymal Stem Cells (MSC) for preparing biocartilage. Thanks to their capacity of proliferation and differentiation, MSC appear to be currently one of the most promising ways of obtaining cartilage cells. The conjunctive tissue of the umbilical cord or Wharton’s jelly (WJ) is an abundant and promising source of MSC for clinical applications. They present immunomodulatory properties and have a higher proliferation potential than MSC from adult tissues. All these advantages enable this source to represent a virtually inexhaustible source of stem cells especially for allogeneic TE therapies. However, immunomodulatory properties of WJ-MSC must be precisely defined as they will be used in an allogeneic clinical context as an Advanced Therapy Medicinal Product (ATMP) according to the European regulation (1394/2007).

The ATYPiCAL project, which comprises 4 complementary teams and a spin off, is the subject of funding project with the agency ANR-PRCE 2015. The innovative proposed strategy relies on a stratified scaffold and consists in using MSC from Wharton’s Jelly (WJ-MSC), extemporaneously embedded into an Alginate/Hyaluronic Acid (Alg/HA) layer guiding cells to chondrogenic differentiation then associated to the NanoM1 medical device (ARTiOS NanoMed) inducing subchondral bone regeneration. An important concern is the use of allogeneic MSC in tissue engineering. The originalities of this present project are:
(i) To perform a technological transfer from research laboratories (UMR 7365 and “Laboratoire d’Immunologie de la Transplantation” of CEA) towards the hospital (UTCT). The transfer consists in adjusting MSC isolation, production and conservation methods developed by UMR 7365 lab in clinical grade conditions at the UTCT. The MSC amplification will be done until the 1st passage and will be followed by a quality control developed by the UMR 7365 and “Laboratoire d’Immunologie de la Transplantation” (CEA) (self-renewal and phenotype stability, genetic stability and cell ageing, differentiation potential, immunomodulation properties). In order to produce clinical grade WJ-MSC in compliance with the current French regulations in the area of Advanced Therapy Medicinal Product (ATMP), the cell culture will take place in a culture medium enriched by human platelet growth factors in order to be free from the presence of animal proteins.
(ii) To use an innovative stratified scaffold and to investigate the construction impact onto chondrogenic differentiation in vitro and in vivo.
(iii) To characterize the immunomodulatory properties of WJ-MSC during chondrogenic differentiation in both in vitro and in vivo studies with functional tests at each step.
(iv) To check, in vitro, that immune response currently observed in an allogeneic context does not negatively interfere with chondrogenic differentiation.
The expected results should allow assessing the feasibility and validating the clinical grade amplification technique as well as obtaining precisely characterized and secured MSC. This project will eventually diversify the ATMP field in France implementing an essential skill to therapeutic progress.

Project coordinator

Madame Danièle Bensoussan (Bioingénerie Moléculaire Cellulaire et Thérapeutique - FR3209)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


BMCT - FR 3209 Bioingénerie Moléculaire Cellulaire et Thérapeutique - FR3209
IMoPA Ingénierie Moléculaire et Physiopathologie Articulaire
CEA-DSV-IMETI Service de Recherches en Hemato-Immunologie
INSERM UMR1260 Osteoarticular and Dental Regenerative Nanomedicine

Help of the ANR 621,031 euros
Beginning and duration of the scientific project: November 2015 - 48 Months

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