Lara Longobardi

Regenerative effects of transplanted mesenchymal stem cells in fracture healing.

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat nonunions. The use of MSC to treat fractures is attractive for several reasons. First, MSCs would be implementing conventional reparative process that seems to be defective or protracted. Secondly, the effects of MSCs treatment would be needed only for relatively brief duration of reparation. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation, and their role in modulating the injury‐related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time‐ and dose‐dependent and, it is exclusively CXCR4‐dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro‐computed tomography and histological studies. Transplanting CMV‐Cre‐R26R‐Lac Z‐MSC, we found that MSCs engrafted within the callus endosteal niche. Using MSCs from BMP‐2‐Lac Z mice genetically modified using a bacterial artificial chromosome system to be β‐gal reporters for bone morphogenic protein 2 (BMP‐2) expression, we found that MSCs contributed to the callus initiation by expressing BMP‐2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow design of novel MSC‐based therapies to treat fractures. STEM CELLS 2009;27:1887–1898

Role of mesenchymal stem cells in regenerative medicine: application to bone and cartilage repair

Background: Mesenchymal stem cells (MSC) are multipotent cells with the ability to differentiate into mesenchyme-derived cells including osteoblasts and chondrocytes. Objective: To provide an overview and expert opinion on the in vivo ability of MSC to home into tissues, their regenerative properties and potential applications for cell-based therapies to treat bone and cartilage disorders. Methods: Data sources including the PubMed database, abstract booklets and conference proceedings were searched for publications pertinent to MSC and their properties with emphasis on the in vivo studies and clinical use in cartilage and bone regeneration and repair. The search included the most current information possible. Conclusion: MSC can migrate to injured tissues and some of their reparative properties are mediated by paracrine mechanisms including their immunomodulatory actions. MSC possess a critical potential in regenerative medicine for the treatment of skeletal diseases, such as osteoarthritis or fracture healing failure, where treatments are partially effective or palliative.

Mesenchymal stem cells at the intersection of cell and gene therapy

Importance of the field : Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secrete bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases. Areas covered in this review: Findings demonstrating the immunomodulation, homing and paracrine activities of MSCs followed by a summary of the current research utilizing MSCs as a vector for gene therapy, focusing on skeletal disorders, but also cardiovascular disease, ischemic damage and cancer. What the reader will gain: MSCs are a possible therapy for many diseases, especially those related to the musculoskeletal system, as a standalone treatment, or in combination with factors that enhance the abilities of these cells to migrate, survive or promote healing through anti-inflammatory and immunomodulatory effects, differentiation, angiogenesis or delivery of cytolytic or anabolic agents. Take home message: Genetically-modified MSCs are a promising area of research that would be improved by focusing on the biology of MSCs that could lead to identification of the natural and engrafting MSC-niche and a consensus on how to isolate and expand MSCs for therapeutic purposes.

Mesenchymal Stem Cells Expressing Insulin‐like Growth Factor‐I (MSCIGF) Promote Fracture Healing and Restore New Bone Formation in Irs1 Knockout Mice: Analyses of MSCIGF Autocrine and Paracrine Regenerative Effects

Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow‐derived MSCs expressing insulin‐like growth factor‐I (MSCIGF), to promote these seed‐and‐soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSCIGF or traceable MSCIGF‐Lac‐Z were transplanted into wild‐type or insulin‐receptor‐substrate knockout (Irs1−/−) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (μCT), and histological analyses. We found that systemically transplanted MSCIGF through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF‐I adapted the response of transplanted MSCIGF to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF‐I‐induced osteoglastogenesis in MSCs was dependent of an intact IRS1‐PI3K signaling. Furthermore, using Irs1−/− mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF‐I on MSC restored the fracture new bone formation and promoted the occurrence of a well‐organized callus that bridged the gap. A callus that was basically absent in Irs1−/− left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSCIGF in fracture repair and potentially to treat nonunions. STEM CELLS 2011;29:1537–1548

Cartilage disorders: potential therapeutic use of mesenchymal stem cells.

Chondrogenesis is a well-orchestrated process driven by chondroprogenitors that undergo to condensation, proliferation and chondrocyte differentiation. Because cartilage lacks regenerative ability, treatments for cartilage diseases are primarily palliative. Adult bone marrow contains a reservoir of mesenchymal stem cells (MSC) with in vitro and in vivo potential of becoming cartilage. To optimize the potential therapeutic use of MSC in cartilage disorders, we need to understand the mechanisms by which growth factors determine their chondrogenic potential. Insulin-like growth factors (IGFs) play a central role in chondrogenesis as indicated by the severe growth failure observed in animals carrying null mutations of Igfs and Igf1R genes. We have found that IGF-I has potent chondrogenic effects in MSC. Effects are similar to transforming growth factor-Beta (TGF-Beta). Insulin-like growth factor binding protein-3 (IGFBP-3), well characterized as IGF carrier, has intrinsic bioactivities that are independent of IGF binding. IGFBP-3 levels are increased in degenerative cartilage diseases such as osteoarthritis. We have demonstrated that IGFBP-3 has IGF-independent growth inhibitory effects in chondroprogenitors. We now show that IGFBP-3 induces MSC apoptosis and antagonizes TGF-Beta chondroinductive effects in chondroprogenitors. Understanding IGF-I chondroinductive and IGFBP-3 chondroinhibitory effects would provide critical information to optimize the therapeutic use of MSC in cartilage disorders.

Subcellular localization of IRS-1 in IGF-I-mediated chondrogenic proliferation, differentiation and hypertrophy of bone marrow mesenchymal stem cells.

Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1− / − mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.

TGF-β Type II Receptor/MCP-5 Axis: At the Crossroad between Joint and Growth Plate Development

Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.

Joint TGF-β type II receptor-expressing cells: ontogeny and characterization as joint progenitors.

TGF-β type II receptor (Tgfbr2) signaling plays an essential role in joint-element development. The Tgfbr2(PRX-1KO) mouse, in which the Tgfbr2 is conditionally inactivated in developing limbs, lacks interphalangeal joints and tendons. In this study, we used the Tgfbr2-β-Gal-GFP-BAC mouse as a LacZ/green fluorescent protein (GFP)-based read-out to determine: the spatial and temporally regulated expression pattern of Tgfbr2-expressing cells within joint elements; their expression profile; and their slow-cycling labeling with bromodeoxyuridine (BrdU). Tgfbr2-β-Gal activity was first detected at embryonic day (E) 13.5 within the interphalangeal joint interzone. By E16.5, and throughout adulthood, Tgfbr2-expressing cells clustered in a contiguous niche that comprises the groove of Ranvier and the synovio-entheseal complex including part of the perichondrium, the synovium, the articular cartilage superficial layer, and the tendons entheses. Tgfbr2-expressing cells were found in the synovio-entheseal complex niche with similar temporal pattern in the knee, where they were also detected in meniscal surface, ligaments, and the synovial lining of the infrapatellar fat pad. Tgfbr2-β-Gal-positive cells were positive for phospho-Smad2, signifying that the Tgfbr2 reporter was accurate. Developmental-stage studies showed that Tgfbr2 expression was in synchrony with expression of joint-morphogenic genes such as Noggin, GDF5, Notch1, and Jagged1. Prenatal and postnatal BrdU-incorporation studies showed that within this synovio-entheseal-articular-cartilage niche most of the Tgfbr2-expressing cells labeled as slow-proliferating cells, namely, stem/progenitor cells. Tgfbr2-positive cells, isolated from embryonic limb mesenchyme, expressed joint progenitor markers in a time- and TGF-β-dependent manner. Our studies provide evidence that joint Tgfbr2-expressing cells have anatomical, ontogenic, slow-cycling trait and in-vivo and ex-vivo expression profiles of progenitor joint cells.

Stachydrine ameliorates high‐glucose induced endothelial cell senescence and SIRT1 downregulation

Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high‐glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high‐glucose (30 mM) for different times. Dose‐dependent (0.001–1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high‐glucose induced EC growth arrest and senescence. Indeed, co‐treatment with high‐glucose and stachydrine for 48 h kept the percentage of EC in the G0/G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal‐laser scanning microscopy revealed that stachydrine also blocked the high‐glucose induced upregulation of p16INK4A and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high‐glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases. J. Cell. Biochem. 114: 2522–2530, 2013.

Use of Glycol Chitosan Modified by 5β-cholanic Acid Nanoparticles for the Sustained Release of Proteins during Murine Embryonic Limb Skeletogenesis

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol chitosan-5beta-cholanic acid conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission electron microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% released after 48 h. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age E13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology.