Autologous Marrow Mesenchymal Stem Cell Driving Bone Regeneration in a Rabbit Model of Femoral Head Osteonecrosis.

Osteonecrosis of the femoral head (ONFH) is a progressive degenerative disease that ultimately requires a total hip replacement. Mesenchymal stromal/stem cells (MSCs), particularly the ones isolated from bone marrow (BM), could be promising tools to restore bone tissue in ONFH. Here, we established a rabbit model to mimic the pathogenic features of human ONFH and to challenge an autologous MSC-based treatment. ON has been originally induced by the synergic combination of surgery and steroid administration. Autologous BM-MSCs were then implanted in the FH, aiming to restore the damaged tissue. Histological analyses confirmed bone formation in the BM-MSC treated rabbit femurs but not in the controls. In addition, the model also allowed investigations on BM-MSCs isolated before (ON-BM-MSCs) and after (ON+BM-MSCs) ON induction to dissect the impact of ON damage on MSC behavior in an affected microenvironment, accounting for those clinical approaches foreseeing MSCs generally isolated from affected patients. BM-MSCs, isolated before and after ON induction, revealed similar growth rates, immunophenotypic profiles, and differentiation abilities regardless of the ON. Our data support the use of ON+BM-MSCs as a promising autologous therapeutic tool to treat ON, paving the way for a more consolidated use into the clinical settings.

Potency Biomarker Signature Genes from Multiparametric Osteogenesis Assays: Will cGMP Human Bone Marrow Mesenchymal Stromal Cells Make Bone?

In skeletal regeneration approaches using human bone marrow derived mesenchymal stromal cells (hBM-MSC), functional evaluation before implantation has traditionally used biomarkers identified using fetal bovine serum-based osteogenic induction media and time courses of at least two weeks. However, emerging pre-clinical evidence indicates donor-dependent discrepancies between these ex vivo measurements and the ability to form bone, calling for improved tests. Therefore, we adopted a multiparametric approach aiming to generate an osteogenic potency assay with improved correlation. hBM-MSC populations from six donors, each expanded under clinical-grade (cGMP) conditions, showed heterogeneity for ex vivo growth response, mineralization and bone-forming ability in a murine xenograft assay. A subset of literature-based biomarker genes was reproducibly upregulated to a significant extent across all populations as cells responded to two different osteogenic induction media. These 12 biomarkers were also measurable in a one-week assay, befitting clinical cell expansion time frames and cGMP growth conditions. They were selected for further challenge using a combinatorial approach aimed at determining ex vivo and in vivo consistency. We identified five globally relevant osteogenic signature genes, notably TGF-ß1 pathway interactors; ALPL, COL1A2, DCN, ELN and RUNX2. Used in agglomerative cluster analysis, they correctly grouped the bone-forming cell populations as distinct. Although donor #6 cells were correlation slope outliers, they contrastingly formed bone without showing ex vivo mineralization. Mathematical expression level normalization of the most discrepantly upregulated signature gene COL1A2, sufficed to cluster donor #6 with the bone-forming classification. Moreover, attenuating factors causing genuine COL1A2 gene down-regulation, restored ex vivo mineralization. This suggested that the signature gene had an osteogenically influential role; nonetheless no single biomarker was fully deterministic whereas all five signature genes together led to accurate cluster analysis. We show proof of principle for an osteogenic potency assay providing early characterization of primary cGMP-hBM-MSC cultures according to their donor-specific bone-forming potential.

cGMP-compliant transportation conditions for a prompt therapeutic use of marrow mesenchymal stromal/stem cells.

We recently described conditions for safe 18-h manufacturer-to-patient transportation of freshly harvested hBM-MSC expanded under cGMP protocols using human platelet lysate (hPL), that allowed prompt use as an advanced therapeutic medicinal product. Here we outline important considerations when comparing different transportation conditions, highlighting that although cell transportation may involve a reduction in viability, this did not undermine the ultimate bone-forming regenerative potential of the cGMP-hBM-MSC population.

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 Ewing's 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.

Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration.

Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/ß-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/ß-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/ß-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18 h away showed prompt adhesion to HA/ß-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.

Delayed marrow infusion in mice enhances hematopoietic and osteopoietic engraftment by facilitating transient expansion of the osteoblastic niche.

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24 hours compared with 6 hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.

Transplanted murine long-term repopulating hematopoietic cells can differentiate to osteoblasts in the marrow stem cell niche.

Bone marrow transplantation (BMT) can give rise to donor-derived osteopoiesis in mice and humans; however, the source of this activity, whether a primitive osteoprogenitor or a transplantable marrow cell with dual hematopoietic and osteogenic potential, has eluded detection. To address this issue, we fractionated whole BM from mice according to cell surface immunophenotype and assayed the hematopoietic and osteopoietic potentials of the transplanted cells. Here, we show that a donor marrow cell capable of robust osteopoiesis possesses a surface phenotype of c-Kit(+) Lin(-) Sca-1(+) CD34(-/lo), identical to that of the long-term repopulating hematopoietic stem cell (LTR-HSC). Secondary BMT studies demonstrated that a single marrow cell able to contribute to hematopoietic reconstitution in primary recipients also drives robust osteopoiesis and LT hematopoiesis in secondary recipients. These findings indicate that LTR-HSC can give rise to progeny that differentiate to osteoblasts after BMT, suggesting a mechanism for prompt restoration of the osteoblastic HSC niche following BM injury, such as that induced by clinical BMT preparative regimens. An understanding of the mechanisms that regulate this differentiation potential may lead to novel treatments for disorders of bone as well as methods for preserving the integrity of endosteal hematopoietic niches.

Mesenchymal stromal/stem cells markers in the human bone marrow.

BACKGROUND AIMS: Mesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation. METHODS: To characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression. RESULTS: Microscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers. CONCLUSIONS: Targeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.

Cytokine-induced osteopoietic differentiation of transplanted marrow cells.

Transplantation of whole bone marrow (BMT) leads to engraftment of both osteoprogenitor cells and hematopoietic cells; however, the robust osteopoietic chimerism seen early after BMT decreases with time. Using our established murine model, we demonstrate that a post-BMT regimen of either granulocyte-colony stimulating factor, growth hormone, parathyroid hormone, or stem cell factor each stimulates greater donor osteoblast chimerism at 4 months posttransplantation than saline-treated controls and approximates the robust osteopoietic chimerism seen early after BMT; however, only growth hormone led to significantly more donor-derived osteocytes than controls. Importantly, there were no adverse hematologic consequences of the different treatments. Our data demonstrate that these cytokines can stimulate the differentiation of transplanted donor marrow cells into the osteopoietic lineage after BMT. Post-BMT cytokine therapy may generate durable osteopoietic engraftment, which should lead to sustained clinical benefit and render BMT more applicable to bone disorders.

Osteopoietic engraftment after bone marrow transplantation: effect of inbred strain of mice.

OBJECTIVE: Transplantable osteoprogenitors, as well as hematopoietic progenitors, reside in bone marrow. We previously reported the first clinical trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. Although the patients demonstrated striking clinical benefits after transplantation, measured osteopoietic engraftment was low and did not seem to be durable. Therefore, we sought an animal model, which closely reflects the clinical experience, to facilitate development of strategies to improve the efficiency of osteoprogenitor engraftment after BMT. MATERIALS AND METHODS: We transplanted unfractionated bone marrow cells from green fluorescent protein-transgenic mice into lethally irradiated recipients in four combinations of inbred mouse strains: from C57BL/6 into C57BL/6 (C-C), from C57BL/6 into FVB/N (C-F), from FVB/N into C57BL/6 (F-C), and from FVB/N into FVB/N (F-F). At 2 weeks after transplantation, we assessed donor hematopoietic and osteopoietic engraftment by flow cytometry, using a novel mean fluorescence assay, and by immunohistochemical staining for green fluorescent protein. RESULTS: Hematopoietic reconstitution by donor cells was complete in all four combinations. Although osteopoietic engraftment of the transplanted cells was also documented in all the four groups, the magnitude of osteopoietic engraftment differed markedly among the strains where F-F > C-F > F-C > C-C. CONCLUSION: Our findings indicate that the genetic background of inbred mouse strains affects efficiency of osteopoietic engraftment after BMT. Thus, the murine strain must be considered when comparing experimental outcomes. Moreover, comparing the genetic variation among murine strains may lend insight into the factors governing osteopoietic differentiation of transplanted marrow cells.

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.

"In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells.

BACKGROUND: The stromal vascular fraction (SVF) is a heterogeneous cell population derived from the adipose tissue. There is still a lack of information concerning the characterization of the cell subpopulations constituting the SVF as well as its mesenchymal and haematopoietic potential. Furthermore there are great variations in its phenotypical characterization. METHODS: Composition of SVF was investigated by FACS analysis, cytological and "in vitro" assays. We studied CD34+ population by combining FACS with human CFC (colony-forming-cell haematopoietic assay). The endothelial fraction was investigated by quantifying the co-expression of specific markers (CD146, CD105, CD31 and UEA-1). Mesenchymal potential was assessed by CFU-F assay and cultured AT-MSC were characterized by a 5-color FACS analysis. The multipotent differentiation potential (osteogenic, adipogenic and chondrogenic) was investigated both at cellular and molecular level. RESULTS: We identified in the SVF two CD34+ populations with a marked difference in the intensity of antigen expression, the majority of the cells expressing CD34 at low intensity. Moreover, two CD146+ cell populations were clearly distinguishable in the SVF:a CD146 dim accounting for 9.9% of the total SVF cells and a CD146+ bright cell population accounting for about 39.3%. The frequency of CFC clones was comparable with the one reported for peripheral blood. Endothelial cells account for about 7.7% of the SVF cells. AT-MSC differenced in the osteogenic adipogenic and chondrogenic lineage. CONCLUSION: The SVF is not a homogeneous cell population, and its final composition could be influenced both by the flow cytometric technique analysis and the SVF extraction steps. The CFU-F frequency in the SVF was 1/4880, a value about seven times greater than the data reported for bone marrow. The antigenic profile of AT-MSC was comparable with bone-marrow derived MSC. AT-MSC were able to differentiate along the osteogenic adipogenic and chondrogenic lineages. The data here reported, further contribute to the characterization of SVF, a tissue providing an alternative as a source of MSC for clinical applications.

Post-transplant Kaposi sarcoma originates from the seeding of donor-derived progenitors.

Kaposi sarcoma (KS) is a vascular tumor that can develop in recipients of solid tissue transplants as a result of either primary infection or reactivation of a gammaherpesvirus, the KS- associated herpesvirus, also known as human herpesvirus-8 (HHV-8). We studied whether HHV-8 and the elusive KS progenitor cells could be transmitted from the donor through the grafts. We used a variety of molecular, cytogenetic, immunohistochemical and immunofluorescence methods to show that the HHV-8-infected neoplastic cells in post-transplant KS from five of eight renal transplant patients harbored either genetic or antigenic markers of their matched donors. These data suggest the use of donor-derived HHV-8-specific T cells for the control of post-transplant KS.

Severe pancytopenia and hemophagocytosis after HHV-8 primary infection in a renal transplant patient successfully treated with foscarnet.

We report the occurrence of human herpesvirus (HHV)-8 primary infection in an adult male kidney recipient. Four months after transplantation, the patient developed visceral Kaposi sarcoma, and 1 month later he presented with progressive and severe peripheral cytopenia, in the presence of a normocellular or hypercellular bone marrow (BM) with hemophagocytosis. HHV-8 was the sole pathogen detected by polymerase chain reaction either in the serum or in the BM. HHV-8 latent nuclear antigen was detected in immature progenitor cells from the BM. Immunosuppressive therapy was reduced, and the patient was treated with foscarnet for 2 weeks, leading to a dramatic normalization of blood cell counts, concomitantly with the disappearance of HHV-8 viremia. At the end of antiviral therapy, the patient received chemotherapy, and Kaposi sarcoma regressed in 2 months. Severe peripheral cytopenia may be a posttransplant complication after HHV-8 infection, for which treatment with foscarnet seems appropriate.

HHV-8 infection in the transplantation setting: a concern only for solid organ transplant patients?

Early epidemiologic studies have shown an increased risk of Kaposi sarcoma (KS) in recipients of solid organ transplants, while KS is exceptional in the setting of autologous and allogeneic bone marrow (BM) and peripheral blood stem cell (PBSC) transplant patients. The recent discovery of human herpesvirus 8 (HHV-8) as the necessary etiologic agent of KS has stimulated studies to assess whether KS is the result of HHV-8 transmission from the donor or of reactivation of a pre-existing HHV-8 infection in the recipient host. An association of HHV-8 infection with lymphoid neoplasias has also been observed, identifying this herpesvirus as a possible causal agent of some Epstein-Barr virus negative post-transplant lymphoproliferative diseases. The recent description of non-neoplastic complications associated with HHV-8 reactivation after autologous PBSC transplantation, has raised concerns about the spectrum of diseases potentially associated with this herpesvirus, even in the setting of BM and or PBSC transplantation. The issue of HHV-8 transmission with the grafts, the problems inherent with the diagnosis and monitoring of active viral infection, the need for prevention and treatment of the clinical consequences of HHV-8 primary infection and reactivation cannot be underestimated, at least in areas endemic for HHV-8 infection.