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1.
Biotechnol Bioeng ; 118(2): 979-991, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33205831

RESUMO

Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with the maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.


Assuntos
Reatores Biológicos , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Linhagem Celular , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia
2.
Curr Diab Rep ; 18(12): 133, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30343423

RESUMO

PURPOSE OF REVIEW: Ever since the reprogramming of human fibroblasts to induced pluripotent stem cells (hiPSCs), scientists have been trying to determine if hiPSCs can give rise to progeny akin to native terminally differentiated cells as human embryonic stem cells (hESCs) do. Many different somatic cell types have been successfully reprogrammed via a variety of methods. In this review, we will discuss recent studies comparing hiPSCs and hESCs and their ability to differentiate to desired cell types as well as explore diabetes disease models. RECENT FINDINGS: Both somatic cell origin and the reprogramming method are important to the epigenetic state of the hiPSCs; however, genetic background contributes the most to differences seen between hiPSCs and hESCs. Based on our review of the relevant literature, hiPSCs display differences compared to hESCs, including a higher propensity for specification toward particular cell types based on memory retained from the somatic cell of origin. Moreover, hiPSCs provide a unique opportunity for creating diabetes disease models.


Assuntos
Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Células Secretoras de Insulina/patologia , Modelos Biológicos , Diferenciação Celular , Humanos
3.
J Biol Eng ; 11: 21, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28680477

RESUMO

Recent advances in the expansion and directed pancreatogenic differentiation of human pluripotent stem cells (hPSCs) have intensified efforts to generate functional pancreatic islet cells, especially insulin-secreting ß-cells, for cell therapies against diabetes. However, the consistent generation of glucose-responsive insulin-releasing cells remains challenging. In this article, we first present basic concepts of pancreatic organogenesis, which frequently serves as a basis for engineering differentiation regimens. Next, past and current efforts are critically discussed for the conversion of hPSCs along pancreatic cell lineages, including endocrine ß-cells and α-cells, as well as exocrine cells with emphasis placed on the later stages of commitment. Finally, major challenges and future directions are examined, such as the identification of factors for in vivo maturation, large-scale culture and post processing systems, cell loss during differentiation, culture economics, efficiency, and efficacy and exosomes and miRNAs in pancreatic differentiation.

4.
Biochem Biophys Res Commun ; 453(2): 243-53, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24971539

RESUMO

Voltage-gated ion channels are transmembrane proteins that regulate electrical excitability in cells and are essential components of the electrically active tissues of nerves, muscle and the heart. Potassium channels are one of the largest subfamilies of voltage sensitive channels and are among the most-studied of the voltage-gated ion channels. Voltage-gated channels can be glycosylated and changes in the glycosylation pattern can affect ion channel function, leading to neurological and neuromuscular disorders and congenital disorders of glycosylation (CDG). Alterations in glycosylation can also be acquired and appear to play a role in development and aging. Recent studies have focused on the impact of glycosylation and sialylation on ion channels, particularly for voltage-gated potassium and sodium channels. The terminal step of sialylation often affects channel activation and inactivation kinetics. The presence of sialic acids on O or N-glycans can alter the gating mechanism and cause conformational changes in the voltage-sensing domains due to sialic acid's negative charges. This manuscript will provide an overview of sialic acids, potassium and sodium channel function, and the impact of sialylation on channel activation and deactivation.


Assuntos
Canais Iônicos/química , Canais Iônicos/metabolismo , Envelhecimento/metabolismo , Animais , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Glicosilação , Crescimento e Desenvolvimento/fisiologia , Humanos , Ativação do Canal Iônico , Canais Iônicos/genética , Modelos Biológicos , Mutação , Ácido N-Acetilneuramínico/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo
5.
Clin Proteomics ; 11(1): 15, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24725457

RESUMO

Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research.

6.
J Proteome Res ; 11(11): 5265-76, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22971049

RESUMO

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.


Assuntos
Proteoma , Acetilação , Animais , Células CHO , Carboidratos/análise , Cromatografia Líquida , Códon , Cricetinae , Cricetulus , Glicosilação , RNA Mensageiro/genética , Espectrometria de Massas em Tandem
7.
Anal Chem ; 83(13): 5296-303, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21591763

RESUMO

Zebrafish (Danio rerio) is a model organism that is used to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development, and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases--chymotrypsin and trypsin--into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid-phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories, which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role that glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.


Assuntos
Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Glicoproteínas/metabolismo , Espectrometria de Massas em Tandem/métodos , Peixe-Zebra/metabolismo , Animais , Peixe-Zebra/embriologia
8.
J Proteome Res ; 10(6): 2777-84, 2011 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-21480662

RESUMO

Protein glycosylation affects cellular functions of the central nervous system (CNS). Its deficiency leads to neurological disorders such as ataxia, paralysis, learning disability, mental retardation, and memory loss. However, the glycoproteins that are responsible for these diseases are not well characterized. In this study, Drosophila melanogaster was used as a model organism to identify the N-glycosylated proteins and N-glycosylation sites of its CNS by means of proteomics. Adult fly heads were digested with chymotrypsin or trypsin and the N-linked glycopeptides were captured using solid phase extraction of N-linked glycopeptides (SPEG) technique followed by mass spectrometry (MS) analysis using LTQ OrbiTrap Velos. Three hundred and thirty new and 147 previously known glycoproteins were identified from 721 uniquely detected peptides that have 740 NXS/T glycosylation sites. The N-glycosylation sites were highly abundant in cell adhesion, ion channel, and ion binding molecules, which are important for nerve maturation, organ development, axon guidance, learning, and memory. Identification of the N-glycosylated sites of these proteins will enhance our knowledge of these proteins and serve as a basis for future studies to address the roles of these proteins in neurological function and disorders. A database for Drosophila N-linked glycopeptides ( http://betenbaugh.jhu.edu/GlycoFly ) has been established in this study as a resource for study of neurological disorders.


Assuntos
Bases de Dados de Proteínas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glicoproteínas/metabolismo , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Proteínas de Drosophila/química , Glicoproteínas/química , Sistemas On-Line , Fragmentos de Peptídeos/química , Proteômica
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