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1.
Cell ; 174(3): 636-648.e18, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30017246

RESUMO

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.


Assuntos
Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Trombopoese/fisiologia , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Humanos , Hidrodinâmica , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Megacariócitos/fisiologia
2.
Physiol Rev ; 99(1): 79-114, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30328784

RESUMO

The discovery of somatic cell nuclear transfer proved that somatic cells can carry the same genetic code as the zygote, and that activating parts of this code are sufficient to reprogram the cell to an early developmental state. The discovery of induced pluripotent stem cells (iPSCs) nearly half a century later provided a molecular mechanism for the reprogramming. The initial creation of iPSCs was accomplished by the ectopic expression of four specific genes (OCT4, KLF4, SOX2, and c-Myc; OSKM). iPSCs have since been acquired from a wide range of cell types and a wide range of species, suggesting a universal molecular mechanism. Furthermore, cells have been reprogrammed to iPSCs using a myriad of methods, although OSKM remains the gold standard. The sources for iPSCs are abundant compared with those for other pluripotent stem cells; thus the use of iPSCs to model the development of tissues, organs, and other systems of the body is increasing. iPSCs also, through the reprogramming of patient samples, are being used to model diseases. Moreover, in the 10 years since the first report, human iPSCs are already the basis for new cell therapies and drug discovery that have reached clinical application. In this review, we examine the generation of iPSCs and their application to disease and development.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Células-Tronco Pluripotentes/classificação , Animais , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Humanos , Fator 4 Semelhante a Kruppel
3.
Nature ; 548(7669): 592-596, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28858313

RESUMO

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.


Assuntos
Modelos Animais de Doenças , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/transplante , Células-Tronco Pluripotentes Induzidas/citologia , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Medicina Regenerativa/métodos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células , Sobrevivência Celular , Neurônios Dopaminérgicos/imunologia , Humanos , Macaca fascicularis , Imageamento por Ressonância Magnética , Masculino , Mesencéfalo/citologia , Movimento , Neostriado/citologia , Neuritos , Doença de Parkinson/etiologia , Doença de Parkinson/fisiopatologia , Tomografia por Emissão de Pósitrons , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo
4.
Int J Mol Sci ; 24(3)2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36768829

RESUMO

Peri-implantitis is a disease that causes the detachment of orthodontic mini-implants. Recently, stress-induced senescent cells have been reported to be involved in various inflammatory diseases. Senescent cell-eliminating drugs, termed "senolytics", can improve the symptoms of such diseases. However, the relationship between peri-implantitis and senescent cells remains unclear. In this study, we evaluated the presence of senescent cells in a rat peri-implantitis model developed with a gum ring. The effect on bone resorption and implant loss was also investigated with and without senolytics (Dasatinib and Quercetin). The number of senescence markers (p19, p21, and p16) was found to increase, and implant detachment occurred in 24 days. After the administration of senolytics, the number of senescence markers decreased and implant detachment was inhibited. This study suggests that senescent cells aggravate peri-implantitis and senolytic administration latently reduces implant loss by inhibiting senescence-related mechanisms.


Assuntos
Reabsorção Óssea , Implantes Dentários , Procedimentos de Ancoragem Ortodôntica , Peri-Implantite , Animais , Ratos , Senescência Celular , Peri-Implantite/tratamento farmacológico , Peri-Implantite/prevenção & controle
5.
Proc Natl Acad Sci U S A ; 114(2): 340-345, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28003464

RESUMO

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.


Assuntos
Arilamina N-Acetiltransferase/metabolismo , Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/fisiologia , Biossíntese de Proteínas/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Fator de Iniciação Eucariótico 4G/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Ligação Proteica/fisiologia , Ribossomos/metabolismo , Proteína SOS1/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia
6.
J Proteome Res ; 18(6): 2535-2544, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31039306

RESUMO

Rapid progress in mass spectrometry (MS) has made comprehensive analyses of the proteome possible, but accurate quantification remains challenging. Isobaric tags for relative and absolute quantification (iTRAQ) is widely used as a tool to quantify proteins expressed in different cell types and various cellular conditions. The quantification precision of iTRAQ is quite high, but the accuracy dramatically decreases in the presence of interference peptides that are coeluted and coisolated with the target peptide. Here, we developed "removal of interference mixture MS/MS spectra (RiMS)" to improve the quantification accuracy of isobaric tag approaches. The presence of spectrum interference is judged by examining the overlap in the elution time of all scanned precursor ions. Removal of this interference decreased protein identification (11% loss) but improved quantification accuracy. Further, RiMS does not require any specialized equipment, such as MS3 instruments or an additional ion separation mode. Finally, we demonstrated that RiMS can be used to quantitatively compare human-induced pluripotent stem cells and human dermal fibroblasts, as it revealed differential protein expressions that reflect the biological characteristics of the cells.


Assuntos
Peptídeos/genética , Proteoma/genética , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Fibroblastos/química , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Peptídeos/química , Peptídeos/isolamento & purificação , Pele/química , Pele/metabolismo , Coloração e Rotulagem
7.
Stem Cells ; 36(10): 1552-1566, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30004605

RESUMO

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552-1566.


Assuntos
Armazenamento de Sangue/métodos , Instabilidade Genômica/genética , Antígenos HLA/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Haplótipos , Antígenos de Histocompatibilidade Classe II , Humanos
8.
Genes Cells ; 22(10): 918-928, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28776863

RESUMO

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity.


Assuntos
Reprogramação Celular/genética , Genes Bacterianos , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Linhagem Celular , Citoesqueleto/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Wolbachia/genética
9.
J Neurosci Res ; 95(9): 1829-1837, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28233934

RESUMO

Patient-specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinson's disease (PD) patients, however, their vulnerability and the transmission of pathological α-Synuclein are possible drawbacks that may prevent PD-specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC-derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α-Synuclein transgenic mice, PDiPSC-derived DA neurons did not cause pathological α-Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD. © 2017 Wiley Periodicals, Inc.


Assuntos
Neurônios Dopaminérgicos/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Doença de Parkinson , Adulto , Idoso , Animais , Feminino , Xenoenxertos , Humanos , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Transplante de Células-Tronco/métodos
10.
Biol Reprod ; 96(6): 1154-1166, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28453617

RESUMO

The mechanisms for human germ cell development have remained largely unknown, due to the difficulty in obtaining suitable experimental materials. The establishment of an in vitro system to reconstitute human germ cell development will thus provide a critical opportunity to understand its mechanisms at a molecular level. It has previously been shown that human induced pluripotent stem cells (hiPSCs) are first induced into incipient mesoderm-like cells (iMeLCs), which are in turn induced into primordial germ-cell like cells (PGCLCs) with gene expression properties similar to early migratory PGCs. Here, we report that the efficiency of PGCLC induction varies among hiPSC clones, and, interestingly, the clonal variations in PGCLC induction efficiency are reflected in the gene expression states of the iMeLCs. Remarkably, the expression levels of EOMES, MIXL1, or T in the iMeLCs are positively correlated with the efficiency of subsequent PGCLC generation, while the expressions of CDH1, SOX3, or FGF2 are negatively correlated. These results indicate that the expression changes of these genes occurring during iMeLC induction are key markers indicative of successful induction of PGCLCs, and furthermore, that hiPSC clones have different properties that influence their responsivity to the iMeLC induction. Our study thus provides important insights into the mechanism of hPGC specification as well as the development of a better strategy for inducing human germ cell fate from PSCs in vitro.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes/fisiologia , Anticorpos , Regulação da Expressão Gênica , Humanos , Cariótipo , Células-Tronco Pluripotentes/classificação , Cromossomos Sexuais
11.
Nucleic Acids Res ; 43(9): e60, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25722368

RESUMO

Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única , Animais , Blastocisto/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
13.
Nature ; 460(7259): 1132-5, 2009 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-19668191

RESUMO

Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.


Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Animais , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Genes myc , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/genética , Linfócitos T/citologia , Transfecção , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
14.
Nat Methods ; 8(5): 409-12, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21460823

RESUMO

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Povo Asiático/genética , Eletroporação , Perfilação da Expressão Gênica , Frequência do Gene , Vetores Genéticos , Antígenos HLA/genética , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Plasmídeos/genética , Doadores de Tecidos
15.
Dent Mater J ; 43(3): 430-436, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38644214

RESUMO

The delayed mucosal healing of tooth extraction sockets in diabetes has few known effective treatment strategies, and its underlying mechanism remains unknown. Senescent cells may play a pivotal role in this delay, given the well-established association between diabetes, senescent cells, and wound healing. Here, we demonstrated an increase in p21- or p16-positive senescent cells in the epithelial and connective tissues of extraction sockets in type 2 diabetic rats compared to those in control rats. Between 7 and 14 days after tooth extraction, a decrease in senescent cells and improvement in re-epithelialization failure were observed in the epithelium, while an increase in senescent cells and persistence of inflammation were observed in the connective tissue. These results suggest that cellular senescence may have been induced by diabetes and contributed to delayed mucosal healing by suppressing re-epithelization and persistent inflammation. These findings provide new targets for treatment using biomaterials, cells, and drugs.


Assuntos
Senescência Celular , Diabetes Mellitus Experimental , Extração Dentária , Cicatrização , Animais , Ratos , Masculino , Diabetes Mellitus Tipo 2/complicações , Alvéolo Dental/patologia , Materiais Biocompatíveis , Mucosa Bucal , Ratos Sprague-Dawley
16.
Regen Ther ; 25: 113-127, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38226057

RESUMO

Introduction: Basic fibroblast growth factor (bFGF, FGF2) and integrin α6ß1 are important for maintaining the pluripotency of human pluripotent stem cells (hPSCs). Although bFGF-integrin binding contributes to biofunctions in cancer cells, the relationship in hPSCs remains unclear. Methods: To investigate the relationship between bFGF and integrin in human induced pluripotent stem cells (hiPSCs), we generated recombinant human bFGF wild-type and mutant proteins, that do not bind to integrin, FGFR, or both. We then cultured hiPSCs with these recombinant bFGF proteins. To evaluate the abilities of recombinant bFGF proteins in maintaining hPSC properties, pluripotent markers, ERK activity, and focal adhesion structure were analyzed through flow cytometry, immunofluorescence (IF), and immunoblotting (IB). Result: We identified an interaction between bFGF and integrin α6ß1 in vitro and in hiPSCs. The integrin non-binding mutant was incapable of inducing the hPSC properties, such as proliferation, ERK activity, and large focal adhesions at the edges of hiPSC colonies. Signaling induced by bFGF-FGFR binding was essential during the first 24 h after cell seeding for maintaining the properties of hPSCs, followed by a shift towards intracellular signaling via the bFGF-integrin interaction. The mixture of the two bFGF mutants also failed to maintain hPSC properties, indicating that bFGF binds to both FGFR and integrin. Conclusion: Our study demonstrates that the integrin-bFGF-FGFR ternary complex maintains the properties of hPSCs via intracellular signaling, providing insights into the functional crosstalk between bFGF and integrins in hiPSCs.

17.
Adv Sci (Weinh) ; 11(26): e2308306, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38685581

RESUMO

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.


Assuntos
Diferenciação Celular , Heparitina Sulfato , Células-Tronco Pluripotentes Induzidas , Laminina , Mesoderma , Diferenciação Celular/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Laminina/metabolismo , Heparitina Sulfato/metabolismo , Desenvolvimento Muscular/fisiologia , Desenvolvimento Muscular/genética , Células Cultivadas
18.
In Vitro Cell Dev Biol Anim ; 60(5): 563-568, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38472720

RESUMO

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Pluripotentes , Humanos , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Criopreservação/métodos , Meios de Cultura/química
19.
In Vitro Cell Dev Biol Anim ; 60(5): 544-554, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38386235

RESUMO

As humans' closest living relatives, chimpanzees offer valuable insights into human evolution. However, technical and ethical limitations hinder investigations into the molecular and cellular foundations that distinguish chimpanzee and human traits. Recently, induced pluripotent stem cells (iPSCs) have emerged as a novel model for functional comparative studies and provided a non-invasive alternative for studying embryonic phenomena. In this study, we generated five new chimpanzee iPSC lines from peripheral blood cells and skin fibroblasts with SeV vectors carrying four reprogramming factors (human OCT3/4, SOX2, KLF4, and L-MYC) and characterized their pluripotency and differentiation potential. We also examined the expression of a human-specific non-coding RNA, HSTR1, which is predicted to be involved in human brain development. Our results show that the chimpanzee iPSCs possess pluripotent characteristics and can differentiate into various cell lineages. Moreover, we found that HSTR1 is expressed in human iPSCs and their neural derivatives but not in chimpanzee counterparts, supporting its possible role in human-specific brain development. As iPSCs are inherently variable due to genetic and epigenetic differences in donor cells or reprogramming procedures, it is essential to expand the number of chimpanzee iPSC lines to comprehensively capture the molecular and cellular properties representative of chimpanzees. Hence, our cells provide a valuable resource for investigating the function and regulation of human-specific transcripts such as HSTR1 and for understanding human evolution more generally.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Fator 4 Semelhante a Kruppel , Pan troglodytes , Animais , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Humanos , Linhagem Celular , Especificidade da Espécie , Fibroblastos/citologia , Fibroblastos/metabolismo , Reprogramação Celular/genética
20.
J Proteome Res ; 12(1): 214-21, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23210603

RESUMO

Proteome analyses of human induced pluripotent stem cells (iPSC) were carried out on a liquid chromatography-tandem mass spectrometry system using meter-scale monolithic silica-C18 capillary columns without prefractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast lysates (4 µg each) were directly injected onto a 200 cm long, 100 µm i.d. monolithic silica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. We identified 98,977 nonredundant tryptic peptides from 9510 proteins (corresponding to 8712 genes), including low-abundance protein groups (such as 329 protein kinases) from triplicate measurements within 10 days. The obtained proteome profiles of the eight cell lysates were categorized into two groups, iPSC and fibroblast, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially modified protein abundance index approach combined with UniProt keyword enrichment analysis revealed that the iPSC group contains more "transcription regulation"-related proteins, while the fibroblast group contained more "transport"-related proteins. Our results indicate that this simplified one-shot proteomics approach with long monolithic columns is advantageous for rapid, deep, sensitive, and reproducible proteome analysis.


Assuntos
Cromatografia Líquida , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Linhagem Celular , Expressão Gênica , Humanos , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Dióxido de Silício/química , Tripsina/genética , Tripsina/isolamento & purificação , Tripsina/metabolismo
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