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1.
Cell Death Dis ; 11(2): 106, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034133

RESUMO

Triple-negative breast cancer (TNBC) is a highly metastatic and deadly disease. TNBC tumors lack estrogen receptor (ERα), progesterone receptor (PR), and HER2 (ErbB2) and exhibit increased glutamine metabolism, a requirement for tumor growth. The G protein-coupled kisspeptin receptor (KISS1R) is highly expressed in patient TNBC tumors and promotes malignant transformation of breast epithelial cells. This study found that TNBC patients displayed elevated plasma kisspeptin levels compared with healthy subjects. It also provides the first evidence that in addition to promoting tumor growth and metastasis in vivo, KISS1R-induced glutamine dependence of tumors. In addition, tracer-based metabolomics analyses revealed that KISS1R promoted glutaminolysis and nucleotide biosynthesis by increasing c-Myc and glutaminase levels, key regulators of glutamine metabolism. Overall, this study establishes KISS1R as a novel regulator of TNBC metabolism and metastasis, suggesting that targeting KISS1R could have therapeutic potential in the treatment of TNBC.

2.
Stem Cells ; 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31904137

RESUMO

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.

3.
Biochem Biophys Res Commun ; 524(1): 50-56, 2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980166

RESUMO

Pulmonary arterial hypertension (PAH) is a rare, but progressive and devastating vascular disease with few treatment options to prevent the advancement to right ventricular dysfunction hypertrophy and failure. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, enhances urinary glucose excretion as well as reduces cardiovascular events and mortality in individuals with type 2 diabetes. While empagliflozin has been reported to lower systemic hypertension due to increased diuresis, the effect of empagliflozin on PAH is unknown. We used monocrotaline (MCT)-treated Sprague-Dawley rats to determine if empagliflozin alters PAH-associated outcomes. Compared to vehicle control, daily empagliflozin administration significantly improved survival in rats with severe MCT-induced PAH. Hemodynamic assessments showed that empagliflozin treatment significantly reduced mean pulmonary artery pressure, right ventricular systolic pressure, and increased pulmonary acceleration time. Empagliflozin treatment resulted in reduced right ventricular hypertrophy and fibrosis. Histological and molecular assessments of lung vasculature revealed significantly reduced medial wall thickening and decreased muscularization of pulmonary arterioles after empagliflozin treatment compared to vehicle-treated rats. In summary, SGLT2 inhibition with empagliflozin lowered mortality, reduced right ventricle systolic pressure, and attenuated maladaptive pulmonary remodeling in MCT-induced PAH. Clinical studies evaluating the efficacy of SGLT-2 inhibition should be considered for patients with PAH.

4.
Cell Metab ; 30(4): 609-613, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31477497

RESUMO

Hess et al. quantified circulating aldehyde dehydrogenase-expressing (ALDHhi) cell subsets in people with T2DM given either empagliflozin (EMPA) or placebo. EMPA treatment increased circulating pro-angiogenic CD133+ progenitor cells, decreased pro-inflammatory ALDHhi granulocyte precursors, and increased ALDHhi monocytes with M2 polarization. EMPA treatment improved T2DM-associated "regenerative cell depletion" contributing to enhanced vascular health.

5.
Trends Mol Med ; 25(7): 640-655, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31053416

RESUMO

Ischemic cardiovascular complications remain a major cause of mortality in people with type 2 diabetes (T2D). Individuals with T2D may have a reduced ability to revascularize ischemic tissues due to abnormal production of circulating provascular progenitor cells. This 'regenerative cell exhaustion' process is intensified by increasing oxidative stress and inflammation and during T2D progression. Chronic exhaustion may be mediated by changes in the bone marrow microenvironment that dysregulate the wingless related integration site network, a central pathway maintaining the progenitor cell pool. Restoration of vascular regenerative cell production by reducing glucotoxicity with contemporary antihyperglycemic agents, by reducing systemic inflammation postbariatric surgery, or by modulating progenitor cell provascular functions using exosomal manipulation, may provide unique approaches for mitigating ischemic disease.

6.
Cell Metab ; 29(4): 792-794, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30943391

RESUMO

Recently in Nature, Wimmer et al. (2019) reported on the development of a human organoid model of vascular development recapitulating vascular pathology during type 2 diabetes. Integration of these organoids into the vasculature of immunodeficient mice may provide cardiometabolic researchers with a "personalized" platform for novel therapeutic discovery.


Assuntos
Diabetes Mellitus Tipo 2 , Angiopatias Diabéticas , Animais , Humanos , Camundongos , Organoides
7.
Cancers (Basel) ; 11(3)2019 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-30909648

RESUMO

Melanoma is one of the most aggressive types of tumors and exhibits high metastatic potential. Fes-related (FER) kinase is a non-receptor tyrosine kinase that has been implicated in growth and metastasis of various epithelial tumors. In this study, we have examined the role that FER kinase plays in melanoma at the molecular level. FER-depleted melanoma cells exhibit impaired Wnt/ß-catenin pathway activity, as well as multiple proteomic changes, which include decreased abundance of L1-cell adhesion molecule (L1-CAM). Consistent with the pro-metastatic functions of these pathways, we demonstrate that depletion of FER kinase decreases melanoma growth and formation of distant metastases in a xenograft model. These findings indicate that FER is an important positive regulator of melanoma metastasis and a potential target for innovative therapies.

8.
JACC Basic Transl Sci ; 4(1): 98-112, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30847424

RESUMO

Detection of vascular regenerative cell exhaustion is required to combat ischemic complications during type 2 diabetes mellitus (T2D). We used high aldehyde dehydrogenase (ALDH) activity and surface marker co-expression to develop a high-throughput flow cytometry-based assay to quantify circulating proangiogenic and proinflammatory cell content in the peripheral blood of individuals with T2D. Circulating proangiogenic monocytes expressing anti-inflammatory M2 markers were decreased in patients with T2D. Individuals with longer duration of T2D exhibited reduced frequencies of circulating proangiogenic ALDHhiCD34+ progenitor cells with primitive (CD133) and migratory (CXCR4) phenotypes. This approach consistently detected increased inflammatory cell burden and decreased provascular progenitor content in individuals with T2D.

9.
Int J Mol Sci ; 20(4)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795516

RESUMO

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Células Cultivadas , Proteínas do Citoesqueleto/química , Estabilidade Enzimática , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas c-raf/química , Ubiquitinação
10.
Stem Cells ; 37(4): 516-528, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30674070

RESUMO

Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into ß-like cells after near complete ß-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous ß-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of ß-cell phenotype by α-cells, and accelerated ß-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of ß-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.

11.
J Biomed Mater Res A ; 107(3): 571-585, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30390406

RESUMO

Cell-based therapies involving the injection of adipose-derived stem/stromal cells (ASCs) within rationally designed biomaterials are a promising approach for stimulating angiogenesis. With this focus, the current work explored the effects of incorporating integrin-binding RGD or IKVAV peptides within in situ-gelling N-methacrylate glycol chitosan (MGC) hydrogels on the response of encapsulated human ASCs. Initial studies focused on hydrogel characterization to validate that the MGC, MGC-RGD, and MGC-IKVAV hydrogels had similar biomechanical properties. ASC viability following encapsulation and culture under 2% O2 was significantly impaired in the MGC-IKVAV group relative to the MGC and MGC-RGD groups. In contrast, sustained viability, along with enhanced cell spreading and metabolic activity were observed in the MGC-RGD group. Investigation of angiogenic transcription suggested that the incorporation of the peptide groups did not substantially alter the pro-angiogenic gene expression profile of the encapsulated ASCs after 7 days of culture under 2% O2. Consistent with the in vitro findings, preliminary in vivo characterization following subcutaneous implantation into NOD/SCID mice showed that ASC retention was enhanced in the MGC-RGD hydrogels relative to the MGC-IKVAV group at 14 days. Further, the encapsulated ASCs in the MGC and MGC-RGD groups promoted murine CD31+ endothelial cell recruitment to the peri-implant region. Overall, the results indicate that the MGC-RGD and MGC hydrogels are promising platforms for ASC delivery, and suggest that strategies that support long-term ASC viability can augment in vivo angiogenesis through paracrine mechanisms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 571-585, 2019.

12.
Cell Rep ; 25(9): 2524-2536.e4, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30485817

RESUMO

Human multipotent stromal cells (hMSCs) are one of the most versatile cell types used in regenerative medicine due to their ability to respond to injury. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSCs is donor specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSCs are lengthy, costly, and low throughput. Therefore, a high-throughput quantitative proteomics assay was developed to screen ß cell regenerative potency of donor-derived hMSC lines. Using proteomics, we identified 16 proteins within hMSC conditioned media that effectively identify ß cell regenerative hMSCs. This protein signature was validated using human islet culture assay, ELISA, and the potency was confirmed by recovery of hyperglycemia in STZ-treated mice. Herein, we demonstrated that quantitative proteomics can determine sample-specific protein signatures that can be used to classify previously uncharacterized hMSC lines for ß cell regenerative clinical applications.


Assuntos
Células Secretoras de Insulina/metabolismo , Células-Tronco Multipotentes/metabolismo , Proteômica/métodos , Regeneração , Adulto , Idoso , Animais , Índice de Massa Corporal , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Multipotentes/efeitos dos fármacos , Reprodutibilidade dos Testes , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Máquina de Vetores de Suporte , Doadores de Tecidos , Adulto Jovem
13.
Blood Adv ; 2(21): 2798-2810, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30355579

RESUMO

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with recurrent mutations that occur in cancer-initiating cells. There is a need to understand how driver mutations influence clonal evolution of leukemia. The E26-transformation-specific (ETS) transcription factors PU.1 and Spi-B (encoded by Spi1 and Spib) execute a critical role in B-cell development and serve as complementary tumor suppressors. Here, we used a mouse model to conditionally delete Spi1 and Spib genes in developing B cells. These mice developed B-ALL with a median time to euthanasia of 18 weeks. We performed RNA and whole-exome sequencing (WES) on leukemias isolated from Mb1-CreΔPB mice and identified single nucleotide variants (SNVs) in Jak1, Jak3, and Ikzf3 genes, resulting in amino acid sequence changes. Jak3 mutations resulted in amino acid substitutions located in the pseudo-kinase (R653H, V670A) and in the kinase (T844M) domains. Introduction of Jak3 T844M into Spi1/Spib-deficient precursor B cells was sufficient to promote proliferation in response to low IL-7 concentrations in culture, and to promote proliferation and leukemia-like disease in transplanted mice. We conclude that mutations in Janus kinases represent secondary drivers of leukemogenesis that cooperate with Spi1/Spib deletion. This mouse model represents a useful tool to study clonal evolution in B-ALL.


Assuntos
Janus Quinase 1/genética , Janus Quinase 3/genética , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/transplante , Proliferação de Células , Modelos Animais de Doenças , Interleucina-7/farmacologia , Janus Quinase 1/química , Janus Quinase 3/química , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Receptores de Interleucina-7/metabolismo , Deleção de Sequência , Transativadores/química
15.
Stem Cells Dev ; 27(20): 1449-1461, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30039749

RESUMO

Islet regeneration is stimulated after transplantation of human umbilical cord blood (UCB) hematopoietic progenitor cells with high aldehyde dehydrogenase (ALDH)-activity into NOD/SCID mice with streptozotocin (STZ)-induced ß cell ablation. ALDHhi progenitor cells represent a rare subset within UCB that will require expansion without the loss of islet regenerative functions for use in cell therapies. ALDHhi cells efficiently expand (>70-fold) under serum-free conditions; however, high ALDH-activity is rapidly diminished during culture coinciding with emergence of a committed megakaryocyte phenotype CD41+/CD42+/CD38+. ALDH-activity is also the rate-limiting step in retinoic acid (RA) production, a potent driver of hematopoietic differentiation. We have previously shown that inhibition of RA production during 9-day cultures, using diethylaminobenzaldehyde (DEAB) treatment, enhanced the expansion of ALDHhi cells (>20-fold) with vascular regenerative paracrine functions. Herein, we sought to determine if DEAB-treatment also expanded ALDHhi cells that retain islet regenerative function following intrapancreatic transplantation into hyperglycemic mice. After DEAB-treatment, expanded ALDHhi cell subset was enriched for CD34+/CD38- expression and demonstrated enhanced myeloid multipotency in vitro compared to the ALDHlo cell subset. Unfortunately, DEAB-treated ALDHhi cells did not support islet regeneration after transplantation. Conversely, expanded ALDHlo cells from DEAB-treated conditions reduced hyperglycemia, and increased islet number and cell proliferation in STZ-induced hyperglycemic NOD/SCID mice. DEAB-treated ALDHlo cells were largely committed to a CD41+/CD42+ megakaryocyte phenotype. Collectively, this study provides preliminary evidence that committed cells of the megakaryocyte-lineage support endogenous islet regeneration and/or function, and the retention of high ALDH-activity did not coincide with islet regenerative function after expansion under serum-free culture conditions.


Assuntos
Aldeído Desidrogenase/genética , Células Secretoras de Insulina/metabolismo , Regeneração/genética , Tretinoína/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Estreptozocina/farmacologia , Tretinoína/química
16.
Oncotarget ; 9(53): 30034-30052, 2018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-30046386

RESUMO

Breast cancer is a leading cause of cancer mortality. In particular, triple negative breast cancer (TNBC) comprise a heterogeneous group of basal-like tumors lacking estrogen receptor (ERα), progesterone receptor (PR) and HER2 (ErbB2). TNBC represents 15-20% of all breast cancers and occurs frequently in women under 50 years of age. Unfortunately, these patients lack targeted therapy, are typically high grade and metastatic at time of diagnosis. The mechanisms regulating metastasis remain poorly understood. We have previously shown that the kisspeptin receptor, KISS1R stimulates invasiveness of TNBC cells. In this report, we demonstrate that KISS1R signals via the secreted extracellular matrix protein, fibulin-3, to regulate TNBC invasion. We found that the fibulin-3 gene is amplified in TNBC primary tumors and that plasma fibulin-3 levels are elevated in TNBC patients compared to healthy subjects. In this study, we show that KISS1R activation increases fibulin-3 expression and secretion. We show that fibulin-3 regulates TNBC metastasis in a mouse experimental metastasis xenograft model and signals downstream of KISS1R to stimulate TNBC invasion, by activating matrix metalloproteinase 9 (MMP-9) and the MAPK pathway. These results identify fibulin-3 as a new downstream mediator of KISS1R signaling and as a potential biomarker for TNBC progression and metastasis, thus revealing KISS1R and fibulin-3 as novel drug targets in TNBC.

17.
Atherosclerosis ; 275: 196-204, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29945035

RESUMO

Primary cilia are microtubule-based organelles that protrude from the cell surface of many mammalian cell types, including endothelial and epithelial cells, osteoblasts, and neurons. These antennal-like projections enable cells to detect extracellular stimuli and elicit responses via intracellular signaling mechanisms. Primary cilia on endothelial cells lining blood vessels function as calcium-dependent mechanosensors that sense blood flow. In doing so, they facilitate the regulation of hemodynamic parameters within the vascular system. Defects in endothelial primary cilia result in inappropriate blood flow-induced responses and contribute to the development of vascular dysfunctions, including atherosclerosis, hypertension, and aneurysms. This review examines the current understanding of vascular endothelial cilia structure and function and their role in the vascular system. Future directions for primary cilia research and treatments for ciliary-based pathologies are discussed.


Assuntos
Doenças Cardiovasculares/metabolismo , Cílios/metabolismo , Células Endoteliais/metabolismo , Hemodinâmica , Mecanotransdução Celular , Animais , Sinalização do Cálcio , Doenças Cardiovasculares/fisiopatologia , Cílios/ultraestrutura , Células Endoteliais/ultraestrutura , Humanos
18.
Stem Cells Dev ; 27(15): 1062-1075, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29737242

RESUMO

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDHhi), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDHhi cells available for transplantation compared with untreated controls. Newly expanded ALDHhi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDHhi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of ß-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating ß-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDHhi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDHhi cells without the loss of islet regenerative functions.


Assuntos
Benzoatos/farmacologia , Diabetes Mellitus Experimental/terapia , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Estilbenos/farmacologia , Antígeno AC133/genética , Aldeído Desidrogenase/genética , Animais , Antígenos CD34/genética , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Sangue Fetal/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Transplante das Ilhotas Pancreáticas , Camundongos , Tretinoína/farmacologia
19.
Biomaterials ; 159: 146-160, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29324306

RESUMO

A promising strategy for treating peripheral ischemia involves the delivery of stem cells to promote angiogenesis through paracrine signaling. Treatment success depends on cell localization, retention, and survival within the mechanically dynamic intramuscular (IM) environment. Herein we describe an injectable, in situ-gelling hydrogel for the IM delivery of adipose-derived stem/stromal cells (ASCs), specifically designed to withstand the dynamic loading conditions of the lower limb and facilitate cytokine release from encapsulated cells. Copolymers of poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate) diacrylate were used to modulate the properties of methacrylated glycol chitosan hydrogels crosslinked by thermally-initiated polymerization using ammonium persulfate and N,N,N',N'-tetramethylethylenediamine. The scaffolds had an ultimate compressive strain over 75% and maintained mechanical properties during compressive fatigue testing at physiological levels. Rapid crosslinking (<3 min) was achieved at low initiator concentration (5 mM). Following injection and crosslinking within the scaffolds, human ASCs demonstrated high viability (>90%) over two weeks in culture under both normoxia and hypoxia. Release of angiogenic and chemotactic cytokines was enhanced from encapsulated cells under sustained hypoxia, in comparison to normoxic and tissue culture polystyrene controls. When delivered by IM injection in a mouse model of hindlimb ischemia, human ASCs were well retained in the scaffold over 28 days and significantly increased the IM vascular density compared to untreated controls.


Assuntos
Citocinas/metabolismo , Células-Tronco/metabolismo , Tecidos Suporte/química , Tecido Adiposo/citologia , Animais , Células Cultivadas , Feminino , Humanos , Hidrogéis/química , Imuno-Histoquímica , Camundongos , Doença Arterial Periférica/metabolismo , Engenharia Tecidual/métodos
20.
Stem Cells ; 36(5): 723-736, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29377410

RESUMO

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDHhi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDHhi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDHhi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDHhi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDHhi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDHhi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Multipotentes/citologia , Regeneração/fisiologia , Animais , Proliferação de Células/fisiologia , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Multipotentes/transplante , Neovascularização Fisiológica/fisiologia
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