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PubMed | Regenerative Medicine Institute, National Center for Biomedical Engineering Science and CSIC - Institute of Polymer Science and Technology
Type: | Journal: Macromolecular rapid communications | Year: 2015

This communication outlines the advances made in the development of thermoresponsive substrates for human mesenchymal stem cell (hMSC) expansion and subsequent controlled specific and multilineage differentiation from a previous study performed by this group. Previously, the development of an inexpensive and technically accessible method for hMSC expansion and harvesting was reported, using the solvent casting deposition method and thermoresponsive poly(N-isopropylacrylamide). Here, the logical continuation of this work is reported with the multipassage expansion of hMSCs with phenotypic maintenance followed by induced adipogenic, osteogenic, and chondrogenic differentiation. Interestingly, 1 m thick solvent cast films are not only capable of hosting an expanding population of phenotypically preserved hMSCs similar to tissue culture plastic controls, but also the cells detached via temperature control better maintain their ability to differentiate compared to conventionally trypsinized cells. This approach to hMSC expansion and differentiation can be highly attractive to stem cell researchers where clinical therapies have seen a collective deviation away from the employment of animal derived products such as proteolytic trypsin.


PubMed | U.S. Stem Cell Inc, Anupam Hospital and Regenerative Medicine Institute
Type: Journal Article | Journal: Journal of translational medicine | Year: 2016

Stromal vascular fraction (SVF) can easily be obtained from a mini-lipoaspirate procedure of fat tissue. The SVF contains a mixture of cells including ADSCs and growth factors and has been depleted of the adipocyte (fat cell) population. We evaluated the safety and efficacy of administering SVF intra-myocardially into patients with chronic ischemic cardiomyopathy.A total of 28 patients underwent a local tumescent liposuction procedure to remove approximately 60ml of fat tissue. The fat was separated to isolate the SVF and the cells were delivered into the akinetic myocardial scar region using a transendocardial delivery system (MyoCath()) in patients who had experienced a previous myocardial infarct. The subjects were then monitored for adverse events, ejection fraction via echocardiogram and six-minute walk test (6MWT) over a period of 6months.The average EF was 29% at baseline and significantly increased to 35% at both 3 and 6months. Patients walked an average of 349m at baseline and demonstrated a statistically significant improvement at 3 and 6months post treatment of more than 80m.Overall, patients were pleased with the treatment results. More importantly, the procedure demonstrated a strong safety profile with no severe adverse events or complications linked to the therapy. Trial registration NCT01502514 Name of registry: http://www.clinicaltrials.gov URL: https://www.clinicaltrials.gov/ct2/show/NCT01502514?term=adipose+cells+heart&rank=4 Date of registration: December 27, 2011 Date of enrollment: January 2012.


Kramerov A.A.,Regenerative Medicine Institute | Ahmed K.,University of Minnesota | Ljubimov A.V.,Regenerative Medicine Institute | Ljubimov A.V.,University of California at Los Angeles
Journal of Cellular Biochemistry | Year: 2012

Protein kinase CK2 participates in a wide range of cellular events, including the regulation of cellular morphology and migration, and may be an important mediator of angiogenesis. We previously showed that in the retina, CK2 immunolocalizes mostly to vascular endothelium and astrocytes in association with the cytoskeleton. Additionally, CK2 inhibitors significantly reduced retinal neovascularization and stem cell recruitment in the mouse model of oxygen-induced proliferative retinopathy. We have also shown that CK2 and F-actin co-localized in actin stress fibers in microvascular endothelial cells, and that highly specific CK2 inhibitors caused cell rounding in astrocytes and microvascular endothelial cells, which was alleviated by serum that promotes spreading by Rho/Rho-kinase (RhoK) activation of myosin II. Therefore, we examined a possible role of CK2 in the regulation of actin-myosin II-based contractility. Treatment with CK2 inhibitors correlated with disassembly of actomyosin stress fibers and cell shape changes, including cytoplasmic retraction and process formation that were similar to those occurring during astrocyte stellation. Low doses of specific inhibitors of kinases (RhoK and MLCK) that phosphorylate myosin light chain (MLC) enhanced the effect of suboptimal CK2 inhibition on cell shape. Such striking stellation-like alteration was accompanied by decreased level of phospho-MLC, thus implying a CK2 role in regulation of actomyosin cytoskeleton. Our results suggest an important role of CK2 in the control of cell contractility and motility, which may account for suppressing effect of CK2 inhibition on retinal neovascularization. Together, our data implicate protein kinase CK2 for the first time in stellation-like morphological transformation. © 2012 Wiley Periodicals, Inc.


Mizrahi O.,Hebrew University of Jerusalem | Sheyn D.,Regenerative Medicine Institute | Tawackoli W.,Regenerative Medicine Institute | Ben-David S.,Regenerative Medicine Institute | And 8 more authors.
Spine Journal | Year: 2013

Background context: The intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear. Purpose: We sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties. Study design: Nucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells. Methods: Intervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal. Results: The comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa. Conclusions: Based on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration. © 2013 Elsevier Inc. All rights reserved.


McCarthy C.,University College Dublin | Duffy M.M.,University College Dublin | Mooney D.,University College Dublin | James W.G.,University College Dublin | And 3 more authors.
FASEB Journal | Year: 2013

Conjugated linoleic acid (CLA) induces regression of preestablished atherosclerosis in the ApoE-/- mouse. Understanding the mechanisms involved may help in identifying novel pathways associated with the regression of human disease. Animals were administered a 1% cholesterol diet for 12 wk, with 1% CLA supplementation from wk 8 to 12. ApoE-/- mice μeμ only the 1% cholesterol diet for 12 wk were employed as controls. Transcriptomic analysis of mouse aorta showed that many of the components of the IL-10 signaling pathway were modified during CLAinduced regression. Real-time PCR and Western blot analysis showed increased IL-10 receptor expression, phosphorylation of STAT3, and downstream target gene expression in the aorta, alongside an increase in serum IL-10 (79.8±22.4 vs. 41.9±5.5 pg/ml, n=10; P<0.01). CLA-supplementation also increased IL-10 production in bone marrow-derived macrophages (143.6±28.6 vs. 94±5.6 pg/ml, n=5; P<0.05). To explore the mechanisms for altered IL-10 production, we examined the profile of monocyte/macrophage phenotype in the vessel wall, bone marrow, and spleen. CLA increased macrophage polarization toward an anti-inflammatory M2 phenotype in vivo, increasing the population of Ly6Clo monocytes (29 vs. 77±14, n=5, P< 0.05) in the aorta. CLA had similar effects on monocytes/ macrophages differentiated from marrow-derived progenitor cells and on splenocytes. The induction of IL-10 on CLA supplementation in this model may reflect a systemic alteration toward an anti-inflammatory phenotype, which, in turn promotes increased vascular infiltration by Ly6Clo monocytes. These cells may contribute to CLA-induced disease regression. © FASEB.


Krakora D.,University of Wisconsin - Madison | Mulcrone P.,University of Wisconsin - Madison | Meyer M.,University of Wisconsin - Madison | Lewis C.,University of Wisconsin - Madison | And 8 more authors.
Molecular Therapy | Year: 2013

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle. © The American Society of Gene & Cell Therapy.


Zhao M.,Virginia Commonwealth University | Sachs P.C.,Regenerative Medicine Institute | Wang X.,Virginia Commonwealth University | Dumur C.I.,Virginia Commonwealth University | And 10 more authors.
Cancer Biology and Therapy | Year: 2012

Data are accumulating to support a role for adipose-derived mesenchymal stem cells (MSCs) in breast cancer progression; however, to date most studies have relied on adipose MSCs from non-breast sources. There is a particular need to investigate the role of adipose MSCs in the pathogenesis of basal-like breast cancer, which develops at a disproportionate rate in pre-menopausal African-American women with a gain in adiposity. The aim of this study was to better understand how breast adipose MSCs (bMSCs) contribute to the progression of basal-like breast cancers by relying on isogenic HMT-3255 S3 (pre-invasive) and T4-2 (invasive) human cells that upon transplantation into nude mice resemble this tumor subtype. In vitro results suggested that bMSCs may contribute to breast cancer progression in multiple ways. bMSCs readily penetrate extracellular matrix components in part through their expression of matrix metalloproteinases 1 and 3, promote the invasion of T4-2 cells and efficiently chemoattract endothelial cells via a bFGF-independent, VEGF-A-dependent manner. As mixed xenografts, bMSCs stimulated the growth, invasion and desmoplasia of T4-2 tumors, yet these resident stem cells showed no observable effect on the progression of pre-invasive S3 cells. While bMSCs form vessel-like structures within Matrigel both in vitro and in vivo and chemoattract endothelial cells, there appeared to be no difference between T4-2/bMSC mixed xenografts and T4-2 xenografts with regard to intra- or peri-tumoral vascularity. Collectively, our data suggest that bMSCs may contribute to the progression of basal-like breast cancers by stimulating growth and invasion but not vasculogenesis or angiogenesis. © 2012 Landes Bioscience.


Connolly R.,National University of Ireland | O'Brien T.,National University of Ireland | O'Brien T.,Regenerative Medicine Institute | Flaherty G.,National University of Ireland | Flaherty G.,International Medical University
Travel Medicine and Infectious Disease | Year: 2014

Methods A web based search utilising five search terms was employed. The first twenty pages of each search result were screened against 340 variables. Results 224 out of 1091 websites advertised stem cell clinics. 68 eligible sites covering 21 countries were evaluated. The top five clinical indications for stem cell therapy were multiple sclerosis, anti-ageing, Parkinson's disease, stroke and spinal cord injury. Adult, autologous stem cells were the most commonly utilised stem cell, and these were frequently sourced from bone marrow and adipose tissue and administered intravenously. Thirty-four per cent of sites mentioned the number of patients treated while one quarter of clinics provided outcome data. Twenty-nine per cent of clinics had an internationally recognised accreditation. Fifteen per cent of clinics stated that their therapies posed no risk. Eighty-eight per cent of clinics claimed treatment effectiveness, with 16% describing their curative potential. Over 40% of sites did not specify the number or duration of treatments. Fifty-three per cent of clinics requested access to patients' medical records, and 12% recommended patients discuss the proposed therapy with their doctor. No clinic recommended that travellers consult a travel medicine specialist or receive vaccinations prior to their intended travel. One quarter of sites discussed contraindications to treatment, with 41% of sites detailing follow up patient care. -absp Conclusions There is potential for stem cell tourists to receive misleading or deficient information from online stem cell clinics. Both the stem cell tourist and travel medicine practitioner should be educated on the potential risks associated with stem cell clinical services advertised online.Background Stem cell therapies are advertised through online resources which describe a range of treatments with diverse clinical indications. Stem cell tourists may not be aware of the information they should seek when consulting these clinics, or of the potential risks involved. The aim of this study was to characterise the therapies offered by online stem cell clinics. © 2014 Elsevier Ltd. All rights reserved.


Sugrue T.,Regenerative Medicine Institute | Sugrue T.,National University of Ireland | Brown J.A.L.,National University of Ireland | Lowndes N.F.,National University of Ireland | And 2 more authors.
Stem Cells | Year: 2013

The regeneration of the hematopoietic system following total body irradiation is supported by host-derived mesenchymal stromal cells (MSCs) within the bone marrow. The mechanisms used by MSCs to survive radiation doses that are lethal to the hematopoietic system are poorly understood. The DNA damage response (DDR) represents a cohort of signaling pathways that enable cells to execute biological responses to genotoxic stress. Here, we examine the role of the DDR in mediating the resistance of MSCs to ionizing radiation (IR) treatment using two authentic clonal mouse MSC lines, MS5 and ST2, and primary bulk mouse MSCs. We show that multiple DDR mechanisms contribute to the radio-resistance of MSCs: robust DDR activation via rapid γ-H2AX formation, activation of effective S and G2/M DNA damage checkpoints, and efficient repair of IR-induced DNA double-strand breaks. We show that MSCs are intrinsically programmed to maximize survival following IR treatment by expressing high levels of key DDR proteins including ATM, Chk2, and DNA Ligase IV; high levels of the anti-apoptotic, Bcl-2 and Bcl-XL; and low levels of the pro-apoptotic, Bim and Puma. As a result, we demonstrate that irradiated mouse MSCs withstand IR-induced genotoxic stress, continue to proliferate, and retain their capacity to differentiate long-term along mesenchymal-derived lineages. We have shown, for the first time, that the DDR plays key roles in mediating the radioresistance of mouse MSCs which may have important implications for the study and application of MSCs in allogeneic bone marrow transplantation, graft-versus-host disease, and cancer treatment. © AlphaMed Press.


Zandian M.,Regenerative Medicine Institute | Mott K.R.,Regenerative Medicine Institute | Allen S.J.,Regenerative Medicine Institute | Dumitrascu O.,Regenerative Medicine Institute | And 3 more authors.
Gene Therapy | Year: 2011

We previously have described a model of multiple sclerosis (MS) in which constitutive expression of murine interleukin (IL)-2 by herpes simplex virus type 1 (HSV-1) (HSV-IL-2) causes central nervous system (CNS) demyelination in different strains of mice. In the current study, we investigated whether this HSV-IL-2-induced demyelination can be blocked using recombinant viruses expressing different cytokines or by injection of plasmid DNA. We have found that coinfection of HSV-IL-2-infected mice with recombinant viruses expressing IL-12p35, IL-12p40 or IL-12p35IL-12p40 did not block the CNS demyelination, and that coinfection with a recombinant virus expressing interferon (IFN)-γ exacerbated it. In contrast, coinfection with a recombinant virus expressing IL-4 reduced demyelination, whereas coinfection of HSV-IL-2-infected mice with a recombinant HSV-1 expressing the IL-12 heterodimer (HSV-IL-12p70) blocked the CNS demyelination in a dose-dependent manner. Similarly, injection of IL-12p70 DNA blocked HSV-IL-2-induced CNS demyelination in a dose-dependent manner and injection of IL-35 DNA significantly reduced CNS demyelination. Injection of mice with IL-12p35 DNA, IL-12p40 DNA, IL-12p35IL-12p40 DNA or IL-23 DNA did not have any effect on HSV-IL-2-induced demyelination, whereas injection of IL-27 DNA increased the severity of the CNS demyelination in the HSV-IL-2-infected mice. This study demonstrates for the first time that IL-12p70 can block HSV-IL-2-induced CNS demyelination and that IL-35 can also reduce this demyelination, whereas IFN-γ and IL-27 exacerbated the demyelination in the CNS of the HSV-IL-2-infected mice. Our results suggest a potential role for IL-12p70 and IL-35 signaling in the inhibition of HSV-IL-2-induced immunopathology by preventing development of autoaggressive T cells. © 2011 Macmillan Publishers Limited All rights reserved.

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