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Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress.

ABSTRACT: The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated.We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL).With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation.

SUBMITTER: Conforti A

PROVIDER: S-EPMC5363595 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress.

Conforti Antonella A Starc Nadia N Biagini Simone S Tomao Luigi L Pitisci Angela A Algeri Mattia M Sirleto Pietro P Novelli Antonio A Grisendi Giulia G Candini Olivia O Carella Cintia C Dominici Massimo M Locatelli Franco F Bernardo Maria Ester ME

Oncotarget 20161101 47

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated.We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to ...[more]

PMID: 27764806

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Project description:BACKGROUND:Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. METHODS:MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. RESULTS:C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. CONCLUSIONS:C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

Project description:Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.

Project description:BACKGROUND:No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma-related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. METHODS:Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX-PAD or a placebo (n = 7) into the injured gluteus medius muscles. RESULTS:We did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as Week 6 in the treatment-groups. Surprisingly, until Week 26, the low-dose group outperformed the high-dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3 , 150 M: 237.4 ± 27.2 cm3 ]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T-cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement. CONCLUSIONS:Allogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.

Dataset Information

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress.

Publications

Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress.

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