Carlotta Spano

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

BACKGROUND Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

Donor cell–derived osteopoiesis originates from a self-renewing stem cell with a limited regenerative contribution after transplantation

In principle, bone marrow transplantation should offer effective treatment for disorders originating from defects in mesenchymal stem cells. Results with the bone disease osteogenesis imperfecta support this hypothesis, although the rate of clinical improvement seen early after transplantation does not persist long term, raising questions as to the regenerative capacity of the donor-derived mesenchymal progenitors. We therefore studied the kinetics and histologic/anatomic pattern of osteopoietic engraftment after transplantation of GFP-expressing nonadherent marrow cells in mice. Serial tracking of donor-derived GFP(+) cells over 52 weeks showed abundant clusters of donor-derived osteoblasts/osteocytes in the epiphysis and metaphysis but not the diaphysis, a distribution that paralleled the sites of initial hematopoietic engraftment. Osteopoietic chimerism decreased from approximately 30% to 10% by 24 weeks after transplantation, declining to negligible levels thereafter. Secondary transplantation studies provided evidence for a self-renewing osteopoietic stem cell in the marrow graft. We conclude that a transplantable, primitive, self-renewing osteopoietic cell within the nonadherent marrow cell population engrafts in an endosteal niche, like hematopoietic stem cells, and regenerates a significant fraction of all bone cells. The lack of durable donor-derived osteopoiesis may reflect an intrinsic genetic program or exogenous environmental signaling that suppresses the differentiation capacity of the donor stem cells.

Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies

Regenerative medicine relying on cell and gene therapies is one of the most promising approaches to repair tissues. Multipotent mesenchymal stem/stromal cells (MSC), a population of progenitors committing into mesoderm lineages, are progressively demonstrating therapeutic capabilities far beyond their differentiation capacities. The mechanisms by which MSC exert these actions include the release of biomolecules with anti-inflammatory, immunomodulating, anti-fibrogenic, and trophic functions. While we expect the spectra of these molecules with a therapeutic profile to progressively expand, several human pathological conditions have begun to benefit from these biomolecule-delivering properties. In addition, MSC have also been proposed to vehicle genes capable of further empowering these functions. This review deals with the therapeutic properties of MSC, focusing on their ability to secrete naturally produced or gene-induced factors that can be used in the treatment of kidney, lung, heart, liver, pancreas, nervous system, and skeletal diseases. We specifically focus on the different modalities by which MSC can exert these functions. We aim to provide an updated understanding of these paracrine mechanisms as a prerequisite to broadening the therapeutic potential and clinical impact of MSC.

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-Xl), regulated by NF-κB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors. (Pathology Oncology Research Vol 12, No 3, 133–142)

Microglia are less pro‐inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus

Activated microglia, astrogliosis, expression of pro‐inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro‐inflammatory cytokines with the activated morphology of microglia, attributing them a pro‐epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS‐based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine‐treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen‐presenting cell phenotype correlated with the presence of CD4pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL‐1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL‐1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)−9 and 12 while microglia only expressed MMP‐12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350–1362

Mesenchymal progenitors aging highlights a mir-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis

Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR‐196a upregulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence, and an improved osteogenesis linked to a dramatic increase of autocrine basic fibroblast growth factor secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance. Stem Cells 2015;33:939–950

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

Mesenchymal Progenitors Expressing TRAIL Induce Apoptosis in Sarcomas

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo‐sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were cocultured with different osteosarcoma, rhabdomyosarcoma, and Ewings Sarcoma (ES) cell lines assessing viability and caspase‐8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC‐TRAIL on tumor size, apoptosis, and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase‐8 activation starting from 8 hours of coculture with MSC‐TRAIL. When injected into pre‐established ES xenotransplants, MSC‐TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC‐TRAIL could also exert potent antiangiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms. Stem Cells 2015;33:859–869