Alexandra Peister

Sarcoma derived from cultured mesenchymal stem cells

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma.

To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heat-shocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

Chondrogenic differentiation of amniotic fluid-derived stem cells.

For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-β (TGF-β) supplementation, with TGF-β1 producing greater increases than TGF-β3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-β1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-β1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications.

3D imaging of tissue integration with porous biomaterials

Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

Stable transfection of MSCs by electroporation

Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 μs in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neor). After electroporation of 106 cells with 10 μg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 μg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.

Cell sourcing for bone tissue engineering: Amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem cells (AFS) and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-ε-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

Novel object recognition in Apoe-/- mice improved by neonatal implantation of wild-type multipotential stromal cells

Multipotential bone marrow stromal cells (MSCs) from wild-type (Wt) or apolipoprotein E deficient (Apoe(-/-)) mice were implanted into the cerebral ventricles of Apoe(-/-) mice. MSCs from Wt mice continued expressing apoE up to 6 months after implantation and were associated with enhanced novel object recognition and increased microtubule-associated protein 2 (MAP2) immunoreactivity in the dentate gyrus. These data show that MSCs can be used to distinguish developmental from post-developmental effects of a gene knockout and support their therapeutic potential for neurodegenerative diseases.

Transplantation of murine bone marrow stromal cells under the kidney capsule to secrete coagulation factor VIII

Ectopic cell transplantation has been studied as an alternative to whole organ transplantation or as a method to produce secretable proteins for genetic disorders. In this study, bone marrow stromal cells isolated from C57Bl/6 mice were genetically modified to express either lacZ- or B-domain-deleted human factor VIII. In vitro modification of the isolated bone marrow stromal cells was initially performed by transducing increased doses of VSV-G pseudotyped lentiviral vectors expressing lacZ. At a MOI of 25, all of the bone marrow stromal cells were X-gal positive, which maintained their ability to expand and differentiate prior to transplantation into mice. Extremely poor engraftment was observed in the liver, but transplantation of the bone marrow stromal cells expressing lacZ under the kidney capsule resulted in long-term viable X-gal-positive cells for at least 8 weeks (length of study). In vitro expression of human factor VIII was detected in a dose-dependent manner following bone marrow stromal cell with a factor VIII-expressing lentiviral vector. Transplantation of the factor VIII-expressing bone marrow stromal cells under the kidney capsule led to long-term therapeutic expression in the mouse plasma (1–3 ng/ml; n = 4–5 mice/group) for 8 weeks. This study demonstrated that ectopic transplantation of bone marrow stromal cells under the kidney capsule can be effective as a method to express secretable proteins in vivo.

Expansion and angiogenic potential of mesenchymal stem cells from patients with critical limb ischemia.

Background: Critical limb ischemia (CLI) is a life‐ and limb‐threatening condition affecting 1% to 10% of the population with peripheral arterial disease. Traditional revascularization options are not possible for up to 50% of CLI patients, in which case, the use of cellular therapies, such as bone marrow‐derived mesenchymal stem cells (MSCs), hold great promise as an alternative revascularization therapy. However, no randomized, controlled phase 3 trials to date have demonstrated an improvement in limb salvage with cellular therapies. This may be due to poor cell quality (ie, inability to generate a sufficient number of angiogenic MSCs) or to the inadequate retention and viability of MSCs after delivery, or both. Because concerns remain about the expansion and angiogenic potential of autologous MSCs in the CLI population, the objective of this study was to examine the effect of our novel culture media supplement, pooled human platelet lysate (PL), in lieu of the standard fetal bovine serum (FBS), to improve the expansion potential of MSCs from CLI patients. We also characterized the in vitro angiogenic activity of MSCs from the tibia of amputated CLI limbs compared with MSCs from healthy donors. Methods: MSCs were obtained from the tibia of four CLI patients (ISC) and four ISC patients with diabetes mellitus (ISC+DM) undergoing major amputation. Healthy MSCs were aspirated from the iliac crest of four young and healthy donors. MSCs were isolated and expanded in culture with PL or FBS. MSCs from passage 3 to 6 were used for phenotypic marker expression and for adipogenic and osteogenic differentiation and were tested for their in vitro angiogenic activity on human microdermal endothelial cells. In parallel MSCs were cultured to passage 11 for population‐doubling calculations. Results: MSCs from ISC and ISC+DM patients and from healthy patients exhibited appropriate expression of cell surface markers and differentiation capacity. Population doublings were significantly greater for PL‐stimulated compared with FBS‐stimulated MSCs in all groups. Biologically active amounts of angiogens were identified in the secretome of all MSCs without consistent trends among groups. PL expansion did not adversely affect the angiogenic activity of MSCs compared with FBS. The ISC and ISC+DM MSCs demonstrated angiogenic effects on endothelial cells similar to those of healthy and ISC MSCs. Conclusions: PL promotes the rapid expansion of MSCs from CLI and healthy persons. Importantly, MSCs expanded from CLI patients demonstrate the desired angiogenic activity compared with their healthy counterparts. We conclude that autologous MSCs from CLI patients can be sufficiently expanded with PL and be expected to deliver requisite angiogenic effects in vivo. We expect the improved expansion of ISC and ISC+DM with PL to be helpful in improving the successful delivery of autologous MSCs to patients with CLI. Clinical Relevance: Despite the widely held belief that mesenchymal stem cells (MSCs) from patients with multiple vascular comorbidities (eg, diabetes, aged patient) are inferior in quality compared with MSCs from healthy patients, we demonstrate that MSCs from patients with critical limb ischemia have similar angiogenic function in vitro to that of healthy patients and that platelet lysate supplementation of culture media improves the expansion potential of these MSCs. These findings support the use of autologous MSCs to promote angiogenesis and our novel MSC expansion protocol for increasing the number of suitable MSCs from patients with critical limb ischemia for cellular therapy.