Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells.

Background: Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods: In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results: We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR-, CD34-, CD45- phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions: The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.

Fibroblasts from the Human Skin Dermo-Hypodermal Junction are Distinct from Dermal Papillary and Reticular Fibroblasts and from Mesenchymal Stem Cells and Exhibit a Specific Molecular Profile Related to Extracellular Matrix Organization and Modeling.

Human skin dermis contains fibroblast subpopulations in which characterization is crucial due to their roles in extracellular matrix (ECM) biology. This study investigates the properties of fibroblasts localized at the frontier of deep dermis and hypodermis, i.e., dermo-hypodermal junction fibroblasts (F-DHJ), which were compared to intermediate reticular dermis (Fr) and superficial papillary dermis (Fp) fibroblasts. F-DHJ differed from Fr and Fp cells in their wider potential for differentiation into mesodermal lineages and in their absence of contractility when integrated in a three-dimensional dermal equivalent. The transcriptomic profile of F-DHJ exhibited specificities in the expression of genes involved in ECM synthesis-processing and "tissue skeleton" organization. In accordance with transcriptome data, ECM proteins, notably Tenascin C, distributions differed between the reticular dermis and the dermo-hypodermal junction areas, which was documented in normal adult skin. Finally, genome-wide transcriptome profiling was used to evaluate the molecular proximity of F-DHJ with the two dermal fibroblast populations (Fp and Fr) and with the mesenchymal stem cells (MSCs) corresponding to five tissue origins (bone marrow, fat, amnion, chorion, and cord). This comparative analysis classified the three skin fibroblast types, including F-DHJ, as a clearly distinct group from the five MSC sample origins.

Long-term effectiveness of local BM-MSCs for skeletal muscle regeneration: a proof of concept obtained on a pig model of severe radiation burn.

BACKGROUND: Medical management of the severe musculocutaneous radiation syndrome involves surgical intervention with debridement of necrotic tissue. Even when skin excision is replaced by specific plastic surgery, treatment of the muscle radiation injury nonetheless remains difficult, for it involves a massive muscle defect in an unpredictable environment, subject to inflammatory waves weeks to months after irradiation, which delay healing and predispose the patient to the development of fibrous scar tissue. In this study, we investigated the long-term effect of local injections of bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery, to treat muscle necrosis in a large animal model. METHODS: Three months after irradiation to the rump, minipigs were treated by excision of necrotic muscle tissue, vascularized flap surgery, and four injections with or without local autologous BM-MSCs, performed weekly. The quality of the muscle wound healing was examined 1 year post-surgery. RESULTS: The skeletal muscle surgery without MSC treatment led to permanent deposition of collagen 1 and 3, decreased myofiber diameter, failed muscle fiber regeneration, a reduced number of capillaries, and the accumulation of high calcium and fat. In animals treated by surgery and MSC injections, these indicators were substantially better and demonstrated established regeneration. MSC therapy acts at several levels by stimulating growth factors such as VEGF, which is involved in angiogenesis and satellite cell pool maintenance, and creating a macrophage M1/M2 balance. CONCLUSION: Thus, cell therapy using BM-MSCs is an effective and safe way to improve recovery of irradiation-induced skeletal muscle damage without signs of long-term degeneration.

Mesenchymal Stromal Cells Based Therapy in Systemic Sclerosis: Rational and Challenges.

Systemic Sclerosis (SSc) is a rare chronic disease, related to autoimmune connective tissue diseases such as Systemic Lupus Erythematosus and Sjögren's Syndrome. Although its clinical heterogeneity, main features of the disease are: extensive tissue fibrosis with increase matrix deposition in skin and internal organ, microvascular alterations and activation of the immune system with autoantibodies against various cellular antigens. In the diffuse cutaneous scleroderma subtype, the disease is rapidly progressive with a poor prognosis, leading to failure of almost any internal organ, especially lung which is the leading cause of death. Primary trigger is unknown but may involve an immune process against mesenchymal cells in a genetically receptive host. Pathophysiology reveals a pivotal role of fibrosis and inflammation alterations implicating different cell subtypes, cytokines and growth factors, autoantibodies and reactive oxygen species. Despite improvement, the overall survival of SSc patients is still lower than that of other inflammatory diseases. Recommended drugs are agents capable of modulating fibrotic and inflammatory pathways. Cellular therapy has recently emerged as a credible option. Besides autologous hematopoietic stem cell transplantation which demonstrated remarkable improvement, mesenchymal stromal cells (MSCs) represent promising therapeutic candidates. Indeed, these cells possess anti-inflammatory, antiproliferative, antifibrotic, and immunomodulary properties especially by secreting a large panel of bioactive molecules, addressing the most important key points of the SSc. In addition, these cells are very sensitive to their environment and are able to modulate their activity according to the pathophysiological context in which they are located. Autologous or allogeneic MSCs from various sources have been tested in many trials in different auto-immune diseases such as multiple sclerosis, Crohn's disease or systemic lupus erythematosus. They are characterized by a broad availability and no or low acute toxicity. However, few randomized prospective clinical trials were published and their production under ATMP regulatory procedures is complex and time-consuming. Many aspects have still to be addressed to ascertain their potential as well as the potential of their derived products in the management of SSc, probably in association with other therapies.

Autologous Bone Marrow Mesenchymal Stem Cells Improve the Quality and Stability of Vascularized Flap Surgery of Irradiated Skin in Pigs.

Cutaneous radiation syndrome has severe long-term health consequences. Because it causes an unpredictable course of inflammatory waves, conventional surgical treatment is ineffective and often leads to a fibronecrotic process. Data about the long-term stability of healed wounds, with neither inflammation nor resumption of fibrosis, are lacking. In this study, we investigated the effect of injections of local autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs), combined with plastic surgery for skin necrosis, in a large-animal model. Three months after irradiation overexposure to the rump, minipigs were divided into three groups: one group treated by simple excision of the necrotic tissue, the second by vascularized-flap surgery, and the third by vascularized-flap surgery and local autologous BM-MSC injections. Three additional injections of the BM-MSCs were performed weekly for 3 weeks. The quality of cutaneous wound healing was examined 1 year post-treatment. The necrotic tissue excision induced a pathologic scar characterized by myofibroblasts, excessive collagen-1 deposits, and inadequate vascular density. The vascularized-flap surgery alone was accompanied by inadequate production of extracellular matrix (ECM) proteins (decorin, fibronectin); the low col1/col3 ratio, associated with persistent inflammatory nodules, and the loss of vascularization both attested to continued immaturity of the ECM. BM-MSC therapy combined with vascularized-flap surgery provided mature wound healing characterized by a col1/col3 ratio and decorin and fibronectin expression that were all similar to that of nonirradiated skin, with no inflammation, and vascular stability. In this preclinical model, vascularized flap surgery successfully and lastingly remodeled irradiated skin only when combined with BM-MSC therapy. Stem Cells Translational Medicine 2018:569-582.

High proportion of PD-1-expressing CD4+ T cells in adipose tissue constitutes an immunomodulatory microenvironment that may support HIV persistence.

We and others have demonstrated that adipose tissue is a reservoir for HIV. Evaluation of the mechanisms responsible for viral persistence may lead to ways of reducing these reservoirs. Here, we evaluated the immune characteristics of adipose tissue in HIV-infected patients receiving antiretroviral therapy (ART) and in non-HIV-infected patients. We notably sought to determine whether adipose tissue's intrinsic properties and/or HIV induced alteration of the tissue environment may favour viral persistence. ART-controlled HIV infection was associated with a difference in the CD4/CD8 T-cell ratio and an elevated proportion of Treg cells in subcutaneous adipose tissue. No changes in Th1, Th2 and Th17 cell proportions or activation markers expression on T cell (Ki-67, HLA-DR) could be detected, and the percentage of CD69-expressing resident memory CD4+ T cells was not affected. Overall, our results indicate that adipose-tissue-resident CD4+ T cells are not extensively activated during HIV infection. PD-1 was expressed by a high proportion of tissue-resident memory CD4+ T cells in both HIV-infected patients and non-HIV-infected patients. Our findings suggest that adipose tissue's intrinsic immunomodulatory properties may limit immune activation and thus may strongly contribute to viral persistence.

Therapeutic potential of gingival fibroblasts for cutaneous radiation syndrome: comparison to bone marrow-mesenchymal stem cell grafts.

Mesenchymal stem cell (MSC) therapy has recently been investigated as a potential treatment for cutaneous radiation burns. We tested the hypothesis that injection of local gingival fibroblasts (GFs) would promote healing of radiation burn lesions and compared results with those for MSC transplantation. Human clinical- grade GFs or bone marrow-derived MSCs were intradermally injected into mice 21 days after local leg irradiation. Immunostaining and real-time PCR analysis were used to assess the effects of each treatment on extracellular matrix remodeling and inflammation in skin on days 28 and 50 postirradiation. GFs induced the early development of thick, fully regenerated epidermis, skin appendages, and hair follicles, earlier than MSCs did. The acceleration of wound healing by GFs involved rearrangement of the deposited collagen, modification of the Col/MMP/TIMP balance, and modulation of the expression and localization of tenascin-C and of the expression of growth factors (VEGF, EGF, and FGF7). As MSC treatment did, GF injection decreased the irradiation-induced inflammatory response and switched the differentiation of macrophages toward an M2-like phenotype, characterized by CD163(+) macrophage infiltration and strong expression of arginase-1. These findings indicate that GFs are an attractive target for regenerative medicine, for easier to collect, can grow in culture, and promote cutaneous wound healing in irradiation burn lesions.

Repeated autologous bone marrow-derived mesenchymal stem cell injections improve radiation-induced proctitis in pigs.

The management of proctitis in patients who have undergone very-high-dose conformal radiotherapy is extremely challenging. The fibrosis-necrosis, fistulae, and hemorrhage induced by pelvic overirradiation have an impact on morbidity. Augmenting tissue repair by the use of mesenchymal stem cells (MSCs) may be an important advance in treating radiation-induced toxicity. Using a preclinical pig model, we investigated the effect of autologous bone marrow-derived MSCs on high-dose radiation-induced proctitis. Irradiated pigs received repeated intravenous administrations of autologous bone marrow-derived MSCs. Immunostaining and real-time polymerase chain reaction analysis were used to assess the MSCs' effect on inflammation, extracellular matrix remodeling, and angiogenesis, in radiation-induced anorectal and colon damages. In humans, as in pigs, rectal overexposure induces mucosal damage (crypt depletion, macrophage infiltration, and fibrosis). In a pig model, repeated administrations of MSCs controlled systemic inflammation, reduced in situ both expression of inflammatory cytokines and macrophage recruitment, and augmented interleukin-10 expression in rectal mucosa. MSC injections limited radiation-induced fibrosis by reducing collagen deposition and expression of col1a2/col3a1 and transforming growth factor-ß/connective tissue growth factor, and by modifying the matrix metalloproteinase/TIMP balance. In a pig model of proctitis, repeated injections of MSCs effectively reduced inflammation and fibrosis. This treatment represents a promising therapy for radiation-induced severe rectal damage.