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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.
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Anticorpos/imunologia , Epitopos/imunologia , Galactosiltransferases/imunologia , alfa-Galactosidase/imunologia , Animais , Vacinas Anticâncer/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transplante Heterólogo/métodosRESUMO
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.
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Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo/citologia , Adulto , Idoso , Envelhecimento , Ciclo Celular , Células Cultivadas , Humanos , Inflamação , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
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Tecido Adiposo/citologia , Células-Tronco Adultas/metabolismo , Ciclo-Oxigenase 1/metabolismo , Hipertensão Pulmonar/terapia , Transplante de Células-Tronco , Células-Tronco Adultas/citologia , Células-Tronco Adultas/transplante , Animais , Diferenciação Celular , Ciclo-Oxigenase 1/genética , Vetores Genéticos/genética , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Lentivirus/genética , Monocrotalina/toxicidade , Ratos , Ratos Sprague-DawleyRESUMO
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.
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Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Exossomos/genética , Exossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Proteômica/métodos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Exossomos/patologia , Feminino , Humanos , Células MCF-7 , Espectrometria de Massas/métodosRESUMO
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.
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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.
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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.
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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.
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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.
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Neoplasias da Mama/patologia , Células-Tronco Mesenquimais/fisiologia , Microambiente Tumoral , Tecido Adiposo/patologia , Animais , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/fisiologia , Quimiotaxia , Meios de Cultivo Condicionados , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-8/metabolismo , Interleucina-8/fisiologia , Células MCF-7 , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Transplante de Neoplasias , TranscriptomaRESUMO
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.
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Neoplasias , Resposta a Proteínas não Dobradas , Humanos , Transdução de Sinais/genética , Ativação Transcricional , Neovascularização PatológicaRESUMO
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.
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Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Miócitos Cardíacos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Miócitos Cardíacos/metabolismo , Sistema de Condução Cardíaco , Células-Tronco/metabolismo , Genes Controladores do DesenvolvimentoRESUMO
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".
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Lesões do Manguito Rotador , Humanos , Corticosteroides/efeitos adversos , Artroscopia , Injeções , Amplitude de Movimento Articular , Manguito Rotador , Lesões do Manguito Rotador/diagnóstico por imagem , Lesões do Manguito Rotador/tratamento farmacológico , Ombro , Resultado do TratamentoRESUMO
(1) Background: Conclusions of meta-analyses of clinical studies may substantially influence opinions of prospective patients and stakeholders in healthcare. Nineteen meta-analyses of clinical studies on the management of primary knee osteoarthritis (pkOA) with stem cells, published between January 2020 and July 2021, came to inconsistent conclusions regarding the efficacy of this treatment modality. It is possible that a separate meta-analysis based on an independent, systematic assessment of clinical studies on the management of pkOA with stem cells may reach a different conclusion. (2) Methods: PubMed, Web of Science, and the Cochrane Library were systematically searched for clinical studies and meta-analyses of clinical studies on the management of pkOA with stem cells. All clinical studies and meta-analyses identified were evaluated in detail, as were all sub-analyses included in the meta-analyses. (3) Results: The inconsistent conclusions regarding the efficacy of treating pkOA with stem cells in the 19 assessed meta-analyses were most probably based on substantial differences in literature search strategies among different authors, misconceptions about meta-analyses themselves, and misconceptions about the comparability of different types of stem cells with regard to their safety and regenerative potential. An independent, systematic review of the literature yielded a total of 183 studies, of which 33 were randomized clinical trials, including a total of 6860 patients with pkOA. However, it was not possible to perform a scientifically sound meta-analysis. (4) Conclusions: Clinicians should interpret the results of the 19 assessed meta-analyses of clinical studies on the management of pkOA with stem cells with caution and should be cautious of the conclusions drawn therein. Clinicians and researchers should strive to participate in FDA and/or EMA reviewed and approved clinical trials to provide clinically and statistically valid efficacy.
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Osteoartrite do Joelho , Humanos , Articulação do Joelho , Osteoartrite do Joelho/terapia , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto , Células-TroncoRESUMO
BACKGROUND: Recently, the management of musculoskeletal disorders with the patients' own stem cells, isolated from the walls of small blood vessels, which can be found in great numbers in the adipose tissue, has received considerable attention. On the other hand, there are still misconceptions about these adipose-derived regenerative cells (ADRCs) that contain vascular-associated pluripotent stem cells (vaPS cells) in regenerative medicine. METHODS: Based on our previous publications on this topic, we have developed a concept to describe the significance of the ADRCs/vaPS cells in the field of orthobiologics as briefly as possible and at the same time as precisely as possible. RESULTS: The ADRCs/vaPS cells belong to the group of orthobiologics that are based on autologous cells. Because the latter can both stimulate a patient's body's localized self-healing power and provide new cells that can integrate into the host tissue during the healing response when the localized self-healing power is exhausted, this group of orthobiologics appears more advantageous than cell-free orthobiologics and orthobiologics that are based on allogeneic cells. Within the group of orthobiologics that are based on autologous cells, enzymatically isolated, uncultured ADRCs/vaPS cells have several advantages over non-enzymatically isolated cells/microfragmented fat as well as over uncultured bone marrow aspirate concentrate and cultured cells (adipose-derived stem cells, bone marrow-derived mesenchymal stem cells). CONCLUSIONS: The use of ADRCs/vaPS cells can be seamlessly integrated into modern orthopedic treatment concepts, which can be understood as the optimization of a process which-albeit less efficiently-also takes place physiologically. Accordingly, this new safe and effective type of treatment is attractive in terms of holistic thinking and personalized medicine.
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Tecido Adiposo , Regeneração Óssea , Doenças Musculoesqueléticas/terapia , Transplante de Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Células-Tronco Pluripotentes , Medicina Regenerativa , Transplante AutólogoRESUMO
Uncultured, unmodified, autologous, adipose-derived regenerative cells (UA-ADRCs) are a safe and effective treatment option for various musculoskeletal pathologies. However, it is unknown whether the composition of the final cell suspension systematically varies with the subject's individual age, sex, body mass index and ethnicity. UA-ADRCs were isolated from lipoaspirate from n = 232 subjects undergoing elective lipoplasty using the Transpose RT system (InGeneron, Inc.; Houston, TX, USA). The UA-ADRCs were assessed for the number of nucleated cells, cell viability and the number of viable nucleated cells per gram of adipose tissue harvested. Cells from n = 37 subjects were further characterized using four-channel flow cytometry. The present study shows, for the first time, that key characteristics of UA-ADRCs can be independent of the subject's age, sex, BMI and ethnicity. This result has important implications for the general applicability of UA-ADRCs in regeneration of musculoskeletal tissue. Future studies must determine whether the independence of key characteristics of UA-ADRCs of the subject's individual age, sex, BMI and ethnicity only applies to the system used in the present study, or also to others of the more than 25 different experimental methods and commercially available systems used to isolate UA-ADRCs from lipoaspirate that have been described in the literature.
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Etnicidade , Lipectomia , Humanos , Índice de Massa Corporal , Sistemas Automatizados de Assistência Junto ao Leito , Tecido AdiposoRESUMO
Increased vascularization, also known as neoangiogenesis, plays a major role in many cancers, including glioblastoma multiforme (GBM), by contributing to their aggressive growth and metastasis. Although anti-angiogenic therapies provide some clinical improvement, they fail to significantly improve the overall survival of GBM patients. Since various pro-angiogenic mediators drive GBM, we hypothesized that identifying targetable genes that broadly inhibit multiple pro-angiogenic mediators will significantly promote favorable outcomes. Here, we identified TRAF3IP2 (TRAF3-interacting protein 2) as a critical regulator of angiogenesis in GBM. We demonstrated that knockdown of TRAF3IP2 in an intracranial model of GBM significantly reduces vascularization. Targeting TRAF3IP2 significantly downregulated VEGF, IL6, ANGPT2, IL8, FZGF2, PGF, IL1ß, EGF, PDGFRB, and VEGFR2 expression in residual tumors. Our data also indicate that exogenous addition of VEGF partially restores angiogenesis by TRAF3IP2-silenced cells, suggesting that TRAF3IP2 promotes angiogenesis through VEGF- and non-VEGF-dependent mechanisms. These results indicate the anti-angiogenic and anti-tumorigenic potential of targeting TRAF3IP2 in GBM, a deadly cancer with limited treatment options.
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Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.
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Infecções por HIV , Soropositividade para HIV , HIV-1 , Alelos , Sistemas CRISPR-Cas , Infecções por HIV/genética , Soropositividade para HIV/genética , HIV-1/genética , Humanos , Receptores CCR5/genética , Replicação ViralRESUMO
BACKGROUND: Tissue resident mesenchymal stem cells (MSCs) are multipotent, self-renewing cells known for their differentiation potential into cells of mesenchymal lineage. The ability of single cell clones isolated from adipose tissue resident MSCs (ASCs) to differentiate into cells of hematopoietic lineage has been previously demonstrated. In the present study, we investigated if the hematopoietic differentiated (HD) cells derived from ASCs could productively be infected with HIV-1. RESULTS: HD cells were generated by differentiating clonally expanded cultures of adherent subsets of ASCs (CD90+, CD105+, CD45-, and CD34-). Transcriptome analysis revealed that HD cells acquire a number of elements that increase their susceptibility for HIV-1 infection, including HIV-1 receptor/co-receptor and other key cellular cofactors. HIV-1 infected HD cells (HD-HIV) showed elevated p24 protein and gag and tat gene expression, implying a high and productive infection. HD-HIV cells showed decreased CD4, but significant increase in the expression of CCR5, CXCR4, Nef-associated factor HCK, and Vpu-associated factor BTRC. HIV-1 restricting factors like APOBEC3F and TRIM5 also showed up regulation. HIV-1 infection increased apoptosis and cell cycle regulatory genes in HD cells. Although undifferentiated ASCs failed to show productive infection, HIV-1 exposure increased the expression of several hematopoietic lineage associated genes such as c-Kit, MMD2, and IL-10. CONCLUSIONS: Considering the presence of profuse amounts of ASCs in different tissues, these findings suggest the possible role that could be played by HD cells derived from ASCs in HIV-1 infection. The undifferentiated ASCs were non-permissive to HIV-1 infection; however, HIV-1 exposure increased the expression of some hematopoietic lineage related genes. The findings relate the importance of ASCs in HIV-1 research and facilitate the understanding of the disease process and management strategies.
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Tecido Adiposo/citologia , HIV-1/fisiologia , Células-Tronco Hematopoéticas/virologia , Células-Tronco Mesenquimais/virologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Antígenos CD4/biossíntese , Antígenos CD4/genética , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Genes cdc , Proteína do Núcleo p24 do HIV/biossíntese , Proteína do Núcleo p24 do HIV/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Receptores CCR4/biossíntese , Receptores CCR4/genética , Receptores CCR5/biossíntese , Receptores CCR5/genética , Regulação para CimaRESUMO
A certain cell type can be isolated from different organs in the adult body that can differentiate into ectoderm, mesoderm, and endoderm, providing significant support for the existence of a certain type of small, vascular-associated, pluripotent stem cell ubiquitously distributed in all organs in the adult body (vaPS cells). These vaPS cells fundamentally differ from embryonic stem cells and induced pluripotent stem cells in that the latter possess the necessary genetic guidance that makes them intrinsically pluripotent. In contrast, vaPS cells do not have this intrinsic genetic guidance, but are able to differentiate into somatic cells of all three lineages under guidance of the microenvironment they are located in, independent from the original tissue or organ where they had resided. These vaPS cells are of high relevance for clinical application because they are contained in unmodified, autologous, adipose-derived regenerative cells (UA-ADRCs). The latter can be obtained from and re-applied to the same patient at the point of care, without the need for further processing, manipulation, and culturing. These findings as well as various clinical examples presented in this paper demonstrate the potential of UA-ADRCs for enabling an entirely new generation of medicine for the benefit of patients and healthcare systems.
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Tecido Adiposo/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular/fisiologia , Humanos , Medicina Regenerativa/métodosRESUMO
CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.