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1.
J Transl Med ; 21(1): 122, 2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36788520

RESUMO

BACKGROUND: Malfunction of astrocytes is implicated as one of the pathological factors of ALS. Thus, intrathecal injection of healthy astrocytes in ALS can potentially compensate for the diseased astrocytes. AstroRx® is an allogeneic cell-based product, composed of healthy and functional human astrocytes derived from embryonic stem cells. AstroRx® was shown to clear excessive glutamate, reduce oxidative stress, secrete various neuroprotective factors, and act as an immunomodulator. Intrathecal injection of AstroRx® to animal models of ALS slowed disease progression and extended survival. Here we report the result of a first-in-human clinical study evaluating intrathecal injection of AstroRx® in ALS patients. METHODS: We conducted a phase I/IIa, open-label, dose-escalating clinical trial to evaluate the safety, tolerability, and therapeutic effects of intrathecal injection of AstroRx® in patients with ALS. Five patients were injected intrathecally with a single dose of 100 × 106 AstroRx® cells and 5 patients with 250 × 106 cells (low and high dose, respectively). Safety and efficacy assessments were recorded for 3 months pre-treatment (run-in period) and 12 months post-treatment (follow-up period). RESULTS: A single administration of AstroRx® at either low or high doses was safe and well tolerated. No adverse events (AEs) related to AstroRx® itself were reported. Transient AEs related to the Intrathecal (IT) procedure were all mild to moderate. The study demonstrated a clinically meaningful effect that was maintained over the first 3 months after treatment, as measured by the pre-post slope change in ALSFRS-R. In the 100 × 106 AstroRx® arm, the ALSFRS-R rate of deterioration was attenuated from - 0.88/month pre-treatment to - 0.30/month in the first 3 months post-treatment (p = 0.039). In the 250 × 106 AstroRx® arm, the ALSFRS-R slope decreased from - 1.43/month to - 0.78/month (p = 0.0023). The effect was even more profound in a rapid progressor subgroup of 5 patients. No statistically significant change was measured in muscle strength using hand-held dynamometry and slow vital capacity continued to deteriorate during the study. CONCLUSIONS: Overall, these findings suggest that a single IT administration of AstroRx® to ALS patients at a dose of 100 × 106 or 250 × 106 cells is safe. A signal of beneficial clinical effect was observed for the first 3 months following cell injection. These results support further investigation of repeated intrathecal administrations of AstroRx®, e.g., every 3 months. TRIAL REGISTRATION: NCT03482050.


Assuntos
Esclerose Lateral Amiotrófica , Transplante de Células-Tronco Mesenquimais , Humanos , Esclerose Lateral Amiotrófica/terapia , Astrócitos , Injeções Espinhais , Transplante de Células-Tronco Mesenquimais/métodos
2.
Hepatology ; 68(4): 1589-1603, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29394503

RESUMO

Transdifferentiation (TD) is the direct reprogramming of adult cells into cells of alternate fate and function. We have previously shown that liver cells can be transdifferentiated into beta-like, insulin-producing cells through ectopic expression of pancreatic transcription factors (pTFs). However, the efficiency of the process was consistently limited to <15% of the human liver cells treated in culture. The data in the current study suggest that liver-to-pancreas TD is restricted to a specific population of liver cells that is predisposed to undergo reprogramming. We isolated TD-predisposed subpopulation of liver cells from >15 human donors using a lineage tracing system based on the Wnt response element, part of the pericentral-specific promoter of glutamine synthetase. The cells, that were propagated separately, consistently exhibited efficient fate switch and insulin production and secretion in >60% of the cells upon pTF expression. The rest of the cells, which originated from 85% of the culture, resisted TD. Both populations expressed the ectopic pTFs with similar efficiencies, followed by similar repression of hepatic genes. Our data suggest that the TD-predisposed cells originate from a distinct population of liver cells that are enriched for Wnt signaling, which is obligatory for efficient TD. In TD-resistant populations, Wnt induction is insufficient to induce TD. An additional step of chromatin opening enables TD of these cells. CONCLUSION: Liver-to-pancreas TD occurs in defined predisposed cells. These cells' predisposition is maintained by Wnt signaling that endows the cells with the plasticity needed to alter their transcriptional program and developmental fate when triggered by ectopic pTFs. These results may have clinical implications by drastically increasing the efficacy of TD in future clinical uses. (Hepatology 2018).


Assuntos
Linhagem da Célula , Transdiferenciação Celular/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Causalidade , Células Cultivadas , Reprogramação Celular , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pâncreas/citologia , Sensibilidade e Especificidade
3.
Front Med (Lausanne) ; 8: 740071, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34778302

RESUMO

Background: An acute respiratory distress syndrome (ARDS) is caused by the increased amounts of pro-inflammatory cytokines and neutrophil-mediated tissue injury. To date, there is no effective treatment for the ARDS available, while the need for one is growing due to the most severe complications of the current coronavirus disease-2019 (COVID-19) pandemic. The human astrocytes (AstroRx) have shown immunomodulatory properties in the central nervous system (CNS). This study aimed to evaluate the capacity of astrocytes to decrease lung inflammation and to be applied as a treatment therapy in ARDS. Methods: First, we assessed the ability of clinical-grade AstroRx to suppress T-cell proliferation in a mixed lymphocyte reaction test. Next, we tested the therapeutical potential of AstroRx cells in a lipopolysaccharide (LPS)-based ARDS mouse model by injecting AstroRx intravenously (i.v). We determined the degree of lung injury by using a severity scoring scale of 0-2, based on the American Thoracic Society. The scoring measured the presence of neutrophils, fibrin deposits, and the thickening of alveolar walls. The state of inflammation was further assessed by quantifying the immune-cell infiltration to the bronchoalveolar lavage fluid (BALF) and by the presence of proinflammatory cytokines and chemokines in the BALF and serum. Results: We detected that AstroRx cells were capable to suppress T-cell proliferation in vitro after exposure to the mitogen concanavalin A (ConA). In vivo, AstroRx cells were able to lower the degree of lung injury in LPS-treated animals compared with the sham injected animals (P = 0.039). In this study, 30% of AstroRx treated mice showed no lung lesions (responder mice), these mice presented a steady number of eosinophils, T cells, and neutrophils comparable with the level of naïve control mice. The inflammatory cytokines and chemokines, such as TNFα, IL1b, IL-6, and CXCL1, were also kept in check in responder AstroRx-treated mice and were not upregulated as in the sham-injected mice (P < 0.05). As a result, the LPS-treated ARDS mice had a higher survival rate when they were treated with AstroRx. Conclusions: Our results demonstrate that the immunosuppressive activity of AstroRx cells support the application of AstroRx cells as a cell therapy treatment for ARDS. The immunoregulatory activity may also be a part of the mechanism of action of AstroRx reported in the amyotrophic lateral sclerosis (ALS) neurodegenerative disease.

4.
Front Endocrinol (Lausanne) ; 12: 635405, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34025576

RESUMO

Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Dipeptidil Peptidase 4/biossíntese , Integrina alfa1/biossíntese , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Peptídeo C/biossíntese , Diferenciação Celular , Separação Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas
5.
PLoS One ; 9(2): e87812, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24504462

RESUMO

Lineage-specific transcription factors (TFs) display instructive roles in directly reprogramming adult cells into alternate developmental fates, in a process known as transdifferentiation. The present study analyses the hypothesis that despite being fast, transdifferentiation does not occur in one step but is rather a consecutive and hierarchical process. Using ectopic expression of Pdx1 in human liver cells, we demonstrate that while glucagon and somatostatin expression initiates within a day, insulin gene expression becomes evident only 2-3 days later. To both increase transdifferentiation efficiency and analyze whether the process indeed display consecutive and hierarchical characteristics, adult human liver cells were treated by three pancreatic transcription factors, Pdx1, Pax4 and Mafa (3pTFs) that control distinct hierarchical stages of pancreatic development in the embryo. Ectopic expression of the 3pTFs in human liver cells, increased the transdifferentiation yield, manifested by 300% increase in the number of insulin positive cells, compared to each of the ectopic factors alone. However, only when the 3pTFs were sequentially supplemented one day apart from each other in a direct hierarchical manner, the transdifferentiated cells displayed increased mature ß-cell-like characteristics. Ectopic expression of Pdx1 followed by Pax4 on the 2(nd) day and concluded by Mafa on the 3(rd) day resulted in increased yield of transdifferentiation that was associated by increased glucose regulated c-peptide secretion. By contrast, concerted or sequential administration of the ectopic 3pTFs in an indirect hierarchical mode resulted in the generation of insulin and somatostatin co-producing cells and diminished glucose regulated processed insulin secretion. In conclusion transcription factors induced liver to pancreas transdifferentiation is a progressive and hierarchical process. It is reasonable to assume that this characteristic is general to wide ranges of tissues. Therefore, our findings could facilitate the development of cell replacement therapy modalities for many degenerative diseases including diabetes.


Assuntos
Transdiferenciação Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Linhagem da Célula/genética , Transdiferenciação Celular/genética , Criança , Pré-Escolar , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/biossíntese , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Somatostatina/biossíntese , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Adulto Jovem
6.
Cell Reprogram ; 12(6): 655-64, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21108535

RESUMO

Reprogramming adult mammalian cells is an attractive approach for generating cell-based therapies for degenerative diseases, such as diabetes. Adult human liver cells exhibit a high level of developmental plasticity and have been suggested as a potential source of pancreatic progenitor tissue. An instructive role for dominant pancreatic transcription factors in altering the hepatic developmental fate along the pancreatic lineage and function has been demonstrated. Here we analyze whether transcription factors expressed in mature pancreatic ß-cells preferentially activate ß-cell lineage differentiation in liver. NKX6.1 is a transcription factor uniquely expressed in ß-cells of the adult pancreas, its potential role in reprogramming liver cells to pancreatic lineages has never been analyzed. Our results suggest that NKX6.1 activates immature pancreatic markers such as NGN-3 and ISL-1 but not pancreatic hormones gene expression in human liver cells. We hypothesized that its restricted capacity to activate a wide pancreatic repertoire in liver could be related to its incapacity to activate endogenous PDX-1 expression in liver cells. Indeed, the complementation of NKX6.1 by ectopic PDX-1 expression substantially and specifically promoted insulin expression and glucose regulated processed hormone secretion to a higher extent than that of PDX-1 alone, without increasing the reprogrammed cells. This may suggest a potential role for NKX6.1 in promoting PDX-1 reprogrammed cells maturation along the ß-cell-like lineage. By contrast, NKX6.1 repressed PDX-1 induced proglucagon gene expression. The individual and concerted effects of pancreatic transcription factors in adult extra-pancreatic cells, is expected to facilitate developing regenerative medicine approaches for cell replacement therapy in diabetics.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/fisiologia , Transativadores/metabolismo , Adolescente , Adulto , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Criança , Pré-Escolar , Glucagon/genética , Glucagon/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Células Secretoras de Insulina/citologia , Fígado/citologia , Transativadores/genética , Adulto Jovem
7.
Transplantation ; 87(6): 821-4, 2009 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-19300183

RESUMO

BACKGROUND: : Accumulated evidence has shown that insulin producing beta-cells in pancreatic islets have the limited potential to regenerate. Adenoviruses have been widely employed to deliver genes of interest into pancreatic islets. This study was aimed at investigating whether adenovirus infection has any impact on the potential of beta-cell proliferation. METHODS: : Human adenovirus type 5 (Ad5) encoding rat insulin promoter driven reporter genes were used to infect freshly isolated pancreatic islets. Western blotting assays were performed to evaluate the expression and activation of key molecules involved in cell survival and proliferation following Ad5 infection. Immunofluorescence staining was employed to identify proliferating cells after culturing the infected and control islets in the presence of BrdU, an analog of thymidine that can be incorporated into the genome of proliferating cells. RESULTS: : Ad5 infection of the islets resulted in expression and activation of Akt1, a key molecule in the PI3 kinase signaling pathway. Accordingly, a higher frequency of islet cell proliferation was detected in Ad5-infected islets than in control islets. DISCUSSION: : These data suggest adenovirus infection can activate beta-cell survival and proliferation machinery, in particular operating through the PI3K/Akt signaling pathway. This information has significant ramification for the use of adenovirus as a gene delivery vehicle for pancreatic islet cells.


Assuntos
Infecções por Adenovirus Humanos/patologia , Ilhotas Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Adenoviridae , Animais , Divisão Celular , Sobrevivência Celular , Ativação Enzimática , Humanos , Insulina/genética , Células Secretoras de Insulina/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Suínos
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