Cristina Grange

Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

Administration of mesenchymal stem cells (MSCs) improves the recovery from acute kidney injury (AKI). The mechanism may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells, but these factors remain unknown. In the current study, we found that microvesicles derived from human bone marrow MSCs stimulated proliferation in vitro and conferred resistance of tubular epithelial cells to apoptosis. The biologic action of microvesicles required their CD44- and beta1-integrin-dependent incorporation into tubular cells. In vivo, microvesicles accelerated the morphologic and functional recovery of glycerol-induced AKI in SCID mice by inducing proliferation of tubular cells. The effect of microvesicles on the recovery of AKI was similar to the effect of human MSCs. RNase abolished the aforementioned effects of microvesicles in vitro and in vivo, suggesting RNA-dependent biologic effects. Microarray analysis and quantitative real time PCR of microvesicle-RNA extracts indicate that microvesicles shuttle a specific subset of cellular mRNA, such as mRNAs associated with the mesenchymal phenotype and with control of transcription, proliferation, and immunoregulation. These results suggest that microvesicles derived from MSCs may activate a proliferative program in surviving tubular cells after injury via a horizontal transfer of mRNA.

Isolation of Renal Progenitor Cells from Adult Human Kidney

We describe here isolation and characterization of CD133+ cells derived from normal adult human kidney. These cells lacked the expression of hematopoietic markers and expressed PAX-2, an embryonic renal marker, suggesting their renal origin. Renal tissue-derived CD133+ cells and clones of individual cells were capable of expansion and limited self-renewal and differentiated in vitro into epithelial or endothelial cells. On subcutaneous implantation in SCID mice, the undifferentiated cells formed tubular structures expressing renal epithelial markers. At variance, when differentiated in endothelial cells, these cells formed functional vessels. On intravenous injection in SCID mice with glycerol-induced tubulonecrosis, the in vitro expanded renal-derived CD133+ cells homed into the injured kidney and integrated in tubules. We propose that CD133+ cells from kidney represent a multipotent adult resident stem cell population that may contribute to the repair of renal injury.

Microvesicles Released from Human Renal Cancer Stem Cells Stimulate Angiogenesis and Formation of Lung Premetastatic Niche

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.

Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.

Identification of a tumor-initiating stem cell population in human renal carcinomas

The purpose of the present study was to search for the presence of a tumor‐initiating stem cell population in renal carcinomas. Based on the recent identification of mesenchymal stem cells in normal kidneys, we sorted cells expressing the mesenchymal stem cell marker CD105 from 5 human renal carcinomas. Because the CD105+ but not the CD105− population showed enhanced tumorigenicity when injected in severely compromised immunodeficient (SCID) mice, we cloned and characterized CD105+ cells and evaluated their stemness, differentiative ability, and serial tumor generation. Characterization of the phenotype of CD105+ clones revealed several stem cell properties: 1) clonogenic ability, 2) expression of nestin, Nanog, Oct4 stem cell markers, and lack of differentiative epithelial markers, 3) ability to grow in non‐adhesive spheroids, 4) in vitro differentiation into epithelial and endothelial cell types, and 5) generation in vivo of serially transplantable carcinomas containing an undifferentiated CD105+ tumorigenic and a differentiated CD105− nontumorigenic population. In addition, some vessels present in carcinomas generated from CD105+ clones were of human origin, suggesting the capability of tumor‐initiating stem cells to in vivo differentiate also in endothelial cells. In conclusion, we demonstrate that CD105+ cells and clones derived from renal carcinomas were enriched in tumor‐initiating cells with stem characteristics.—Bussolati, B., Bruno, S., Grange, C., Ferrando, U., Camussi, G. Identification of a tumor‐initiating stem cell population in human renal carcinomas. FASEB J. 22, 3696–3705 (2008)

Microvesicles Derived from Human Bone Marrow Mesenchymal Stem Cells Inhibit Tumor Growth

Mesenchymal stem cells (MSCs) have opposite effects on tumor growth, being able either to favor angiogenesis and tumor initiation or to inhibit progression of established tumors. Factors produced by MSCs within the tumor microenvironment may be relevant for their biological effects. Recent studies demonstrated that microvesicles (MVs) are an integral component of inter-cellular communication within the tumor microenvironment. In the present study, we evaluated whether MVs derived from human bone marrow MSCs may stimulate or inhibit in vitro and in vivo growth of HepG2 hepatoma, Kaposis sarcoma, and Skov-3 ovarian tumor cell lines. We found that MVs inhibited cell cycle progression in all cell lines and induced apoptosis in HepG2 and Kaposis cells and necrosis in Skov-3. The observed activation of negative regulators of cell cycle may explain these biological effects. In vivo intra-tumor administration of MVs in established tumors generated by subcutaneous injection of these cell lines in SCID mice significantly inhibited tumor growth. In conclusion, MVs from human MSCs inhibited in vitro cell growth and survival of different tumor cell lines and in vivo progression of established tumors.

Magnetic Resonance Visualization of Tumor Angiogenesis by Targeting Neural Cell Adhesion Molecules with the Highly Sensitive Gadolinium-Loaded Apoferritin Probe

Tumor vessel imaging could be useful in identifying angiogenic blood vessels as well as being a potential predictive marker of antiangiogenic treatment response. We recently reported the expression of the neural cell adhesion molecule (NCAM) in the immature and tumor endothelial cell (TEC) lining vessels of human carcinomas. Exploiting an in vivo model of human tumor angiogenesis obtained by implantation of TEC in Matrigel in severe combined immunodeficiency mice, we aimed to image angiogenesis by detecting the expression of NCAM with magnetic resonance imaging. The imaging procedure consisted of (a) targeting NCAMs with a biotinylated derivative of C3d peptide that is known to have high affinity for these epitopes and (b) delivery of a streptavidin/gadolinium (Gd)-loaded apoferritin 1:1 adduct at the biotinylated target sites. The remarkable relaxation enhancement ability of the Gd-loaded apoferritin system allowed the visualization of TEC both in vitro and in vivo when organized in microvessels connected to the mouse vasculature. Gd-loaded apoferritin displayed good in vivo stability and tolerability. The procedure reported herein may be easily extended to the magnetic resonance visualization of other epitopes suitably targeted by proper biotinylated vectors.

Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery.

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.

Endothelial progenitor cell-derived microvesicles improve neovascularization in a murine model of hindlimb ischemia.

Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast-derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs 0.39±0.03, p < 0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs 13.5±6, p < 0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p < 0.05), suggesting a role of RNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.

Endothelial cell differentiation of human breast tumour stem/progenitor cells

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross‐lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct‐4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere‐derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary‐like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere‐derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3–4 weeks, an epithelial tumour suggesting that in the endothelial‐differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour‐initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.