A New Humanized Mouse Model Mimics Humans in Lacking α-Gal Epitopes and Secreting Anti-Gal Antibodies.

Mice have been used as accepted tools for investigating complex human diseases and new drug therapies because of their shared genetics and anatomical characteristics with humans. However, the tissues in mice are different from humans in that human cells have a natural mutation in the α1,3 galactosyltransferase (α1,3GT) gene and lack α-Gal epitopes on glycosylated proteins, whereas mice and other nonprimate mammals express this epitope. The lack of α-Gal epitopes in humans results in the loss of immune tolerance to this epitope and production of abundant natural anti-Gal Abs. These natural anti-Gal Abs can be used as an adjuvant to enhance processing of vaccine epitopes to APCs. However, wild-type mice and all existing humanized mouse models cannot be used to test the efficacy of vaccines expressing α-Gal epitopes because they express α-Gal epitopes and lack anti-Gal Abs. Therefore, in an effort to bridge the gap between the mouse models and humans, we developed a new humanized mouse model that mimics humans in that it lacks α-Gal epitopes and secretes human anti-Gal Abs. The new humanized mouse model (Hu-NSG/α-Galnull) is designed to be used for preclinical evaluations of viral and tumor vaccines based on α-Gal epitopes, human-specific immune responses, xenotransplantation studies, and in vivo biomaterials evaluation. To our knowledge, our new Hu-NSG/α-Galnull is the first available humanized mouse model with such features.

A novel model to characterize structure and function of BRCA1.

BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1Δ/Δ181-1415 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1Δ/Δ126-136 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1Δ/Δ525-881 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1Δ/Δ872-878 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.

Low expression of galectin-3 is associated with poor survival in node-positive breast cancers and mesenchymal phenotype in breast cancer stem cells.

BACKGROUND: Galectin-3 (Gal3) plays diverse roles in cancer initiation, progression, and drug resistance depending on tumor type characteristics that are also associated with cancer stem cells (CSCs). Recurrence of breast carcinomas may be attributed to the presence of breast CSCs (BCSCs). BCSCs exist in mesenchymal-like or epithelial-like states and the transition between these states endows BCSCs with the capacity for tumor progression. The discovery of a feedback loop with galectins during epithelial-to-mesenchymal transition (EMT) prompted us to investigate its role in breast cancer stemness. METHOD: To elucidate the role of Gal3 in BCSCs, we performed various in vitro and in vivo studies such as sphere-formation assays, Western blotting, flow cytometric apoptosis assays, and limited dilution xenotransplant models. Histological staining for Gal3 in tissue microarrays of breast cancer patients was performed to analyze the relationship of clinical outcome and Gal3 expression. RESULTS: Here, we show in a cohort of 87 node-positive breast cancer patients treated with doxorubicin-based chemotherapy that low Gal3 was associated with increased lymphovascular invasion and reduced overall survival. Analysis of in vitro BCSC models demonstrated that Gal3 knockdown by small hairpin RNA (shRNA) interference in epithelial-like mammary spheres leads to EMT, increased sphere-formation ability, drug-resistance, and heightened aldefluor activity. Furthermore, Gal3negative BCSCs were associated with enhanced tumorigenicity in orthotopic mouse models. CONCLUSIONS: Thus, in at least some breast cancers, loss of Gal3 might be associated with EMT and cancer stemness-associated traits, predicts poor response to chemotherapy, and poor prognosis.

Chemoprevention of colorectal cancer by targeting APC-deficient cells for apoptosis.

Cancer chemoprevention uses natural, synthetic, or biological substances to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. It holds promise for overcoming problems associated with the treatment of late-stage cancers. However, the broad application of chemoprevention is compromised at present by limited effectiveness and potential toxicity. To overcome these challenges, here we developed a new chemoprevention approach that specifically targets premalignant tumour cells for apoptosis. We show that a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of beta-catenin lead to the repression of cellular caspase-8 inhibitor c-FLIP (also known as CFLAR) expression through activation of c-Myc, and that all-trans-retinyl acetate (RAc) independently upregulates tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors and suppresses decoy receptors. Thus, the combination of TRAIL and RAc induces apoptosis in APC-deficient premalignant cells without affecting normal cells in vitro. In addition, we show that short-term and non-continuous TRAIL and RAc treatment induce apoptosis specifically in intestinal polyps, strongly inhibit tumour growth, and prolong survival in multiple intestinal neoplasms C57BL/6J-Apc(Min)/J (Apc(Min)) mice. With our approach, we further demonstrate that TRAIL and RAc induce significant cell death in human colon polyps, providing a potentially selective approach for colorectal cancer chemoprevention by targeting APC-deficient cells for apoptosis.

The impact of statins on biological characteristics of stem cells provides a novel explanation for their pleiotropic beneficial and adverse clinical effects.

Statins reduce atherosclerotic events and cardiovascular mortality. Their side effects include memory loss, myopathy, cataract formation, and increased risk of diabetes. As cardiovascular mortality relates to plaque instability, which depends on the integrity of the fibrous cap, we hypothesize that the inhibition of the potential of mesenchymal stem cells (MSCs) to differentiate into macrophages would help to explain the long known, but less understood "non-lipid-associated" or pleiotropic benefit of statins on cardiovascular mortality. In the present investigation, MSCs were treated with atorvastatin or pravastatin at clinically relevant concentrations and their proliferation, differentiation potential, and gene expression profile were assessed. Both types of statins reduced the overall growth rate of MSCs. Especially, statins reduced the potential of MSCs to differentiate into macrophages while they exhibited no direct effect on macrophage function. These findings suggest that the limited capacity of MSCs to differentiate into macrophages could possibly result in decreased macrophage density within the arterial plaque, reduced inflammation, and subsequently enhance plaque stability. This would explain the non-lipid-associated reduction in cardiovascular events. On a negative side, statins impaired the osteogenic and chondrogenic differentiation potential of MSCs and increased cell senescence and apoptosis, as indicated by upregulation of p16, p53 and Caspase 3, 8, and 9. Statins also impaired the expression of DNA repair genes, including XRCC4, XRCC6, and Apex1. While the effect on macrophage differentiation explains the beneficial side of statins, their impact on other biologic properties of stem cells provides a novel explanation for their adverse clinical effects.

Two sides of the same coin: stem cells in cancer and regenerative medicine.

Multipotent stromal cells (MSCs) derived from bone marrow, adipose tissue, cord blood, and other origins have recently received much attention as potential therapeutic agents with beneficial immunomodulatory and regenerative properties. In their native tissue environment, however, such cells also appear to have essential functions in building and supporting tumor microenvironments, providing metastatic niches, and maintaining cancer hallmarks. Here, we consider the varied roles of these tissue-resident stroma-associated cells, synthesize recent and emerging discoveries, and discuss the role, potential, and clinical applications of MSCs in cancer and regenerative medicine.-Ilmer, M., Vykoukal, J., Recio Boiles, A., Coleman, M., Alt, E. Two sides of the same coin: stem cells in cancer and regenerative medicine.

Intratracheal administration of cyclooxygenase-1-transduced adipose tissue-derived stem cells ameliorates monocrotaline-induced pulmonary hypertension in rats.

The effect of intratracheal administration of cyclooxygenase-1 (COX-1)-modified adipose stem cells (ASCs) on monocrotaline-induced pulmonary hypertension (MCT-PH) was investigated in the rat. The COX-1 gene was cloned from rat intestinal cells, fused with a hemagglutanin (HA) tag, and cloned into a lentiviral vector. The COX-1 lentiviral vector was shown to enhance COX-1 protein expression and inhibit proliferation of vascular smooth muscle cells without increasing apoptosis. Human ASCs transfected with the COX-1 lentiviral vector (ASCCOX-1) display enhanced COX-1 activity while exhibiting similar differentiation potential compared with untransduced (native) ASCs. PH was induced in rats with MCT, and the rats were subsequently treated with intratracheal injection of ASCCOX-1 or untransduced ASCs. The intratracheal administration of ASCCOX-1 3 × 10(6) cells on day 14 after MCT treatment significantly attenuated MCT-induced PH when hemodynamic values were measured on day 35 after MCT treatment whereas administration of untransduced ASCs had no significant effect. These results indicate that intratracheally administered ASCCOX-1 persisted for at least 21 days in the lung and attenuate MCT-induced PH and right ventricular hypertrophy. In addition, vasodilator responses to the nitric oxide donor sodium nitroprusside were not altered by the presence of ASCCOX-1 in the lung. These data emphasize the effectiveness of ASCCOX-1 in the treatment of experimentally induced PH.

Molecular characterization of exosome-like vesicles from breast cancer cells.

BACKGROUND: Membrane vesicles released by neoplastic cells into extracellular medium contain potential of carrying arrays of oncogenic molecules including proteins and microRNAs (miRNA). Extracellular (exosome-like) vesicles play a major role in cell-to-cell communication. Thus, the characterization of proteins and miRNAs of exosome-like vesicles is imperative in clarifying intercellular signaling as well as identifying disease markers. METHODS: Exosome-like vesicles were isolated using gradient centrifugation from MCF-7 and MDA-MB 231 cultures. Proteomic profiling of vesicles using liquid chromatography-mass spectrometry (LC-MS/MS) revealed different protein profiles of exosome-like vesicles derived from MCF-7 cells (MCF-Exo) than those from MDA-MB 231 cells (MDA-Exo). RESULTS: The protein database search has identified 88 proteins in MDA-Exo and 59 proteins from MCF-Exo. Analysis showed that among all, 27 proteins were common between the two exosome-like vesicle types. Additionally, MDA-Exo contains a higher amount of matrix-metalloproteinases, which might be linked to the enhanced metastatic property of MDA-MB 231 cells. In addition, microarray analysis identified several oncogenic miRNA between the two types vesicles. CONCLUSIONS: Identification of the oncogenic factors in exosome-like vesicles is important since such vesicles could convey signals to non-malignant cells and could have an implication in tumor progression and metastasis.

The Circulasome: A Unifying Theory of Flap Vascularity.

Despite the critical roles of nondominant perforators, choke vessels, and direct and indirect linking vessels in flap vascularity, current models of flap perfusion focus on a primary large caliber perforators. The delay phenomenon, microvascularization, neovascularization, and vascular evolution, which depend on smaller caliber vessels, remain unaccounted for. We propose that the "circulasome" consists of the sum of the entire vascular components of a given region, such that the region is supplied by a primary supplying vessel. The circulasome represents one of the indices of flap supply and is proportional to the angiogenic potential of the region and the vascular substrate capable of promoting growth of vascular networks. By accounting for both the primary flap supplying vessel and secondary vascular structures, the circulasome provides a unifying explanation for neovascularization, delay phenomenon, angiosome and perforasome theories, and vascular evolution in flaps.

Analysis of Major and Minor Pedicles in Flap Perfusion by Computational Fluid Dynamics.

Background: Type 2 muscle flaps are characterized by major and minor pedicles, such that the minor pedicle is unreliable, and the major pedicle is a requirement for the success of the flap. The role of the minor pedicle, beyond the decreased caliber and decreased vascular territory in comparison to the major pedicle, is poorly understood. We sought to model the fluid dynamics of a model flap containing a major and minor pedicle to understand differences between the pedicles and the implications on perfusion. Methods: We first generated a computer-assisted design model of a type 2 flap with a major and minor pedicle. We then performed computational fluid dynamics to analyze velocities and flow within the pedicles and flap. Results: In our investigation, we found that the flow velocity within the major pedicle was higher than the minor pedicle, indicative of decreased resistance to flow. Concomitantly, we found decreased pressures within the major pedicle, reflecting decreasing resistance to flow. Interestingly, we found increased kinematic viscosity in flap areas supplied by the minor pedicle, suggesting decreased flow rates and increased resistance. Conclusions: We identified that the major pedicle has increased flow velocity, decreased resistance, and decreased kinematic viscosity, suggesting its dominance in maintaining flap perfusion. Our study also identifies computational fluid dynamics as a powerful tool in studying flap perfusion dynamics.

Nontopical Nitrates in Flap Perfusion and Delay Phenomenon.

Background: Topical nitrates have demonstrated efficacy in improving flap perfusion. However, evidence for nontopical nitrates in modulation of flap perfusion dynamics has yet to be consolidated. Here, we review evidence regarding the use of intravascular, sublingual, and oral nitrates in modulating flap perfusion. Methods: We performed a review of the literature for evidence linking nontopical nitrates and flap perfusion, and included clinical studies, animal studies, and in vitro studies. Results: Evidence suggests that intravascular, sublingual, and oral nitrates exert vasodilatory properties, which may be harnessed for identification of perforators and improved flap perfusion. We also found evidence suggesting nitrates may facilitate ischemic preconditioning while reducing ischemia-reperfusion injury. Conclusions: Nitrates delivered intravascularly, sublingually, or orally may increase flap perfusion and serve as a method for ischemic preconditioning, particularly in the intraoperative setting.

Modulation of Cellular Stemness for Enhanced Fat Grafting.

Low volumetric retention limits the utility of fat grafting. Although inclusion of stem cells and platelet-rich plasma have been proposed to enhance graft retention, accumulating evidence has failed to show a clear benefit. Here, we propose a strategy to pharmacologically enhance stemness of stem and progenitor cell populations in fat grafts to promote increased volume retention and tissue health. We also propose how to integrate stemness-promoting and differentiation-promoting therapies such as platelet-rich plasma, and viability promoting therapies within the common fat grafting workflow to achieve optimal fat grafting results.

Prospective dual role of mesenchymal stem cells in breast tumor microenvironment.

Breast cancer tissue is a heterogeneous cellular milieu comprising cancer and host cells. The interaction between breast malignant and non-malignant cells takes place in breast tumor microenvironment (TM), and has a crucial role in breast cancer progression. In addition to cellular component of TM, it mainly consists of cytokines released by tumor cells. The tumor-tropic capacity of mesenchymal stem cells (MSCs) and their interaction with breast TM is an active area of investigation. In the present communication, the interplay between the breast resident adipose tissue-derived MSCs (B-ASCs) and breast TM was studied. It was found that a distinct subset of B-ASCs display a strong affinity for conditioned media (CM) from two breast cancer cell lines, MDA-MB 231 (MDA-CM) and MCF-7 (MCF-CM). The expressions of several cytokines including angiogenin, GM-CSF, IL-6, GRO-α and IL-8 in MDA-CM and MCF-CM have been identified. Upon functional analysis a crucial role for GRO-α and IL-8 in B-ASCs migration was detected. The B-ASC migration was found to be via negative regulation of RECK and enhanced expression of MMPs. Furthermore, transcriptome analysis showed that migratory subpopulation express both pro- and anti-tumorigenic genes and microRNAs (miRNA). Importantly, we observed that the migratory cells exhibit similar gene and miRNA attributes as those seen in B-ASCs of breast cancer patients. These findings are novel and suggest that in breast cancer, B-ASCs migrate to the proximity of tumor foci. Characterization of the molecular mechanisms involved in the interplay between B-ASCs and breast TM will help in understanding the probable role of B-ASCs in breast cancer development, and could pave way for anticancer therapies.

Percutaneous intraportal application of adipose tissue-derived mesenchymal stem cells using a balloon occlusion catheter in a porcine model of liver fibrosis.

PURPOSE: To investigate the safety and effectiveness of a novel endovascular approach for therapeutic cell delivery using a balloon occlusion catheter in a large animal model of liver fibrosis. MATERIALS AND METHODS: Transcatheter arterial embolization with ethiodized oil (Ethiodol) and ethanol was used to induce liver damage in 11 pigs. Mesenchymal stem cells (MSCs) were harvested from adipose tissue and engineered to express green fluorescent protein (GFP). A balloon occlusion catheter was positioned in the bilateral first-order portal vein branches 2 weeks after embolization to allow intraportal application of MSCs in six experimental animals. MSCs were allowed to dwell for 10 minutes using prolonged balloon inflation. Five control animals received a sham injection of normal saline in a similar fashion. Hepatic venous pressure gradient (HVPG) was measured immediately before necropsy. Specimens from all accessible lobes were obtained with ultrasound-guided percutaneous 18-gauge biopsy 2 hours after cell application. All animals were euthanized within 4 weeks. Fluorescent microscopy was used to assess the presence and distribution of cells. RESULTS: Liver injury and fibrosis were successfully induced in all animals. MSCs (6-10 × 10(7)) were successfully delivered into the portal vein in the six experimental animals. Cell application was not associated with vascular complications. HVPG showed no instances of portal hypertension. GFP-expressing MSCs were visualized in biopsy specimens and were distributed primarily within the sinusoidal spaces; however, 4 weeks after implantation, MSCs could not be identified in histologic specimens. CONCLUSIONS: A percutaneous endovascular approach for cell delivery using a balloon occlusion catheter proved safe for intraportal MSC application in a large animal model of liver fibrosis.

Safety and Efficacy of Autologous Stem Cell Treatment for Facetogenic Chronic Back Pain.

BACKGROUND: Chronic back pain due to facet joint syndrome is a common and debilitating condition. Advances in regenerative medicine have shown that autologous unmodified adipose tissue-derived regenerative cells (ADRC) provide several beneficial effects. These regenerative cells can differentiate into various tissues and exhibit a strong anti-inflammatory potential. ADRCs can be obtained from a small amount of fatty tissue derived from the patient's abdominal fat. METHODS: We report long-term results of 37 patients (age 31-78 years, mean 62.5) suffering from "Facet Joint Syndrome" The pathology was confirmed by clinical, radiological examinations and fluoroscopically guided test injections. Then, liposuction was performed. An amount of 50-100 cc of fat was harvested. To recover stem cells from adipose tissue, we use the CE-certified Transpose RT™ system from InGeneron GmbH. The cells were then injected under fluoroscopic control in the periarticular fat. Follow-up examinations were performed at 1 week, 1 year, and 5 years. RESULTS: Every patient reported improved VAS pain at any follow-up (1 week, 1 year, and 5 years) with ADRCs compared to the baseline. CONCLUSIONS: Our observational data indicate that facet joint syndrome patients treated with unmodified adipose tissue-derived regenerative cells experience improved the quality of life in the long term.

Conversion of Unmodified Stem Cells to Pacemaker Cells by Overexpression of Key Developmental Genes.

Arrhythmias of the heart are currently treated by implanting electronic pacemakers and defibrillators. Unmodified adipose tissue-derived stem cells (ASCs) have the potential to differentiate into all three germ layers but have not yet been tested for the generation of pacemaker and Purkinje cells. We investigated if-based on overexpression of dominant conduction cell-specific genes in ASCs-biological pacemaker cells could be induced. Here we show that by overexpression of certain genes that are active during the natural development of the conduction system, the differentiation of ASCs to pacemaker and Purkinje-like cells is feasible. Our study revealed that the most effective procedure consisted of short-term upregulation of gene combinations SHOX2-TBX5-HCN2, and to a lesser extent SHOX2-TBX3-HCN2. Single-gene expression protocols were ineffective. Future clinical implantation of such pacemaker and Purkinje cells, derived from unmodified ASCs of the same patient, could open up new horizons for the treatment of arrythmias.

Unfolded protein response and angiogenesis in malignancies.

Cellular stress, arising from accumulation of unfolded proteins, occurs frequently in rapidly proliferating cancer cells. This cellular stress, in turn, activates the unfolded protein response (UPR), an interconnected set of signal transduction pathways that alleviate the proteostatic stress. The UPR is implicated in cancer cell survival and proliferation through upregulation of pro-tumorigenic pathways that ultimately promote malignant metabolism and neoangiogenesis. Here, we reviewed mechanisms of signaling crosstalk between the UPR and angiogenesis pathways, as well as transmissible ER stress and the role in tumor growth and development. To characterize differences in UPR and UPR-mediated angiogenesis in malignancy, we employed a data mining approach using patient tumor data from The Cancer Genome Atlas (TCGA). The analysis of TCGA revealed differences in UPR between malignant samples versus their non-malignant counterparts.

Management of partial-thickness rotator cuff tears with autologous adipose-derived regenerative cells is safe and more effective than injection of corticosteroid.

Symptomatic, partial-thickness rotator cuff tears (sPTRCT) are problematic. This study tested the hypothesis that management of sPTRCT with injection of fresh, uncultured, unmodified, autologous, adipose-derived regenerative cells (UA-ADRCs) is safe and more effective than injection of corticosteroid even in the long run. To this end, subjects who had completed a former randomized controlled trial were enrolled in the present study. At baseline these subjects had not responded to physical therapy treatments for at least 6 weeks, and were randomly assigned to receive respectively a single injection of UA-ADRCs (n = 11) or a single injection of methylprednisolone (n = 5). Efficacy was assessed using the ASES Total score, pain visual analogue scale (VAS), RAND Short Form-36 Health Survey and range of motion at 33.2 ± 1.0 (mean ± SD) and 40.6 ± 1.9 months post-treatment. Proton density, fat-saturated, T2-weighted MRI of the index shoulder was performed at both study visits. There were no greater risks connected with injection of UA-ADRCs than those connected with injection of corticosteroid. The subjects in the UA-ADRCs group showed statistically significantly higher mean ASES Total scores than the subjects in the corticosteroid group. The MRI scans at 6 months post-treatment allowed to "watch the UA-ADRCs at work".

Epithelial-mesenchymal transition in breast cancer lines is mediated through PDGF-D released by tissue-resident stem cells.

Epithelial-mesenchymal transition (EMT) generates tumor cells with stem cell properties. The aim of our study was to investigate the effects of adipose tissue-derived stem cells (ASCs) on EMT of cancer cells and to further investigate the mechanisms involved. We demonstrate that conditioned medium from ASCs induces breast cancer cells (4T1) to express mesenchymal markers such as fibronectin, alpha smooth muscle actin and vimentin. Flow cytometry analyses show that ASC-conditioned medium promotes the expansion of CD44high/CD24low cancer stem cells. Soft agar assays using T47D, BT474 and MCF-7 breast cancer cells reveals that ASC conditioned medium promotes the anchorage-independent growth of cancer cells. These effects were inhibited by a neutralizing antibody against platelet-derived growth factor-D (PDGF-D). Furthermore, PDGF-D treated breast cancer cells grow faster in a mouse model, and this effect could be neutralized by a PDGF antibody. In conclusion, our data show that tissue-resident stem cells interact with the cancer microenvironment via PDGF-D, induce EMT in the cancer cells in a paracrine fashion, thereby increasing the number of cancer stem cells and increase tumor growth in a PDGF dependent manner. Our findings shed new light on mechanisms where local tissue-resident stem cells are able to promote the growth of breast cancer cells. Possibly this could open up a novel selective therapeutic strategy targeting EMT pathways and the specific communication between tissue-resident normal stem cell and cancer stem cells, assuming that the blockage of PDGF-D pathways is critical for tumor growth but would not affect normal tissue homeostasis.

Interleukin-8 derived from local tissue-resident stromal cells promotes tumor cell invasion.

The aim of this study is to evaluate the role of adipose tissue resident stromal cells on tumor cell invasion. Our data show that a subpopulation of adipose tissue derived stromal cells expressing Nestin, NG2, α-smooth muscle actin and PDGFR-α migrate toward the cancer cells. Microarray analysis revealed the upregulation of IL-8 in the migrated cells. We demonstrated that stromal cell derived IL-8 promote the invasion and the anchorage-independent growth of cancer cells. We conclude that human breast cancer cells attract a subpopulation of stromal cells that secrete IL-8 to promote tumor cell invasion in a paracrine fashion.