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1.
Nat Protoc ; 19(2): 565-594, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38087082

RESUMO

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Microscopia , Reprodutibilidade dos Testes , Técnicas de Cultura de Células/métodos , Automação
2.
Nature ; 613(7943): 345-354, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36599983

RESUMO

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.


Assuntos
Células-Tronco Pluripotentes Induzidas , Espaço Intracelular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Célula Única , Conjuntos de Dados como Assunto , Interfase , Forma Celular , Mitose , Polaridade Celular , Sobrevivência Celular
3.
Sci Rep ; 11(1): 15845, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349150

RESUMO

We performed a comprehensive analysis of the transcriptional changes occurring during human induced pluripotent stem cell (hiPSC) differentiation to cardiomyocytes. Using single cell RNA-seq, we sequenced > 20,000 single cells from 55 independent samples representing two differentiation protocols and multiple hiPSC lines. Samples included experimental replicates ranging from undifferentiated hiPSCs to mixed populations of cells at D90 post-differentiation. Differentiated cell populations clustered by time point, with differential expression analysis revealing markers of cardiomyocyte differentiation and maturation changing from D12 to D90. We next performed a complementary cluster-independent sparse regression analysis to identify and rank genes that best assigned cells to differentiation time points. The two highest ranked genes between D12 and D24 (MYH7 and MYH6) resulted in an accuracy of 0.84, and the three highest ranked genes between D24 and D90 (A2M, H19, IGF2) resulted in an accuracy of 0.94, revealing that low dimensional gene features can identify differentiation or maturation stages in differentiating cardiomyocytes. Expression levels of select genes were validated using RNA FISH. Finally, we interrogated differences in cardiac gene expression resulting from two differentiation protocols, experimental replicates, and three hiPSC lines in the WTC-11 background to identify sources of variation across these experimental variables.


Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Transcriptoma , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , RNA-Seq
4.
Stem Cell Reports ; 12(5): 1145-1158, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30956114

RESUMO

We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/genética , Actinina/genética , Actinina/metabolismo , Sequência de Aminoácidos , Diferenciação Celular/genética , Linhagem Celular , Reparo do DNA por Junção de Extremidades/genética , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Sarcômeros/metabolismo , Homologia de Sequência de Aminoácidos , Troponina I/genética , Troponina I/metabolismo
5.
J Vis Exp ; (138)2018 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-30199041

RESUMO

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.


Assuntos
Sistemas CRISPR-Cas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Cultivadas , Humanos
6.
Mol Biol Cell ; 28(21): 2854-2874, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28814507

RESUMO

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.


Assuntos
Imunofluorescência/métodos , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco/fisiologia , Sistemas CRISPR-Cas , Linhagem Celular , Marcação de Genes/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
7.
Cell Rep ; 7(4): 1284-97, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24794432

RESUMO

Historically, spontaneous deletions and insertions have provided means to probe germline developmental genetics in Drosophila, mouse and other species. Here, induced pluripotent stem cell (iPSC) lines were derived from infertile men with deletions that encompass three Y chromosome azoospermia factor (AZF) regions and are associated with production of few or no sperm but normal somatic development. AZF-deleted iPSC lines were compromised in germ cell development in vitro. Undifferentiated iPSCs transplanted directly into murine seminiferous tubules differentiated extensively to germ-cell-like cells (GCLCs) that localized near the basement membrane, demonstrated morphology indistinguishable from fetal germ cells, and expressed germ-cell-specific proteins diagnostic of primordial germ cells. Alternatively, all iPSCs that exited tubules formed primitive tumors. iPSCs with AZF deletions produced significantly fewer GCLCs in vivo with distinct defects in gene expression. Findings indicate that xenotransplantation of human iPSCs directs germ cell differentiation in a manner dependent on donor genetic status.


Assuntos
Azoospermia/patologia , Fertilidade/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Túbulos Seminíferos/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pele/citologia , Transplante Heterólogo/métodos
8.
Radiat Res ; 173(4): 494-504, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20334522

RESUMO

Space travel and prolonged bed rest cause bone loss due to musculoskeletal disuse. In space, radiation fields may also have detrimental consequences because charged particles traversing the tissues of the body can elicit a wide range of cytotoxic and genotoxic lesions. The effects of heavy-ion radiation exposure in combination with musculoskeletal disuse on bone cells and tissue are not known. To explore this, normally loaded 16-week-old male C57BL/6 mice were exposed to (56)Fe ions (1 GeV/nucleon) at doses of 0 cGy (sham), 10 cGy, 50 cGy or 2 Gy 3 days before tissue harvest. Additional mice were hindlimb unloaded by tail traction continuously for 1 week to simulate weightlessness and exposed to (56)Fe-ion radiation (0 cGy, 50 cGy, 2 Gy) 3 days before tissue harvest. Despite the short duration of this study, low-dose (10, 50 cGy) irradiation of normally loaded mice reduced trabecular volume fraction (BV/TV) in the proximal tibiae by 18% relative to sham-irradiated controls. Hindlimb unloading together with 50 cGy radiation caused a 126% increase in the number of TRAP(+) osteoclasts on cancellous bone surfaces relative to normally loaded, sham-irradiated controls. Together, radiation and hindlimb unloading had a greater effect on suppressing osteoblastogenesis ex vivo than either treatment alone. In sum, low-dose exposure to heavy ions (50 cGy) caused rapid cancellous bone loss in normally loaded mice and increased osteoclast numbers in hindlimb unloaded mice. In vitro irradiation also was more detrimental to osteoblastogenesis in bone marrow cells that were recovered from hindlimb unloaded mice compared to cells from normally loaded mice. Furthermore, irradiation in vitro stimulated osteoclast formation in a macrophage cell line (RAW264.7) in the presence of RANKL (25 ng/ml), showing that heavy-ion radiation can stimulate osteoclast differentiation even in the absence of osteoblasts. Thus heavy-ion radiation can acutely increase osteoclast numbers in cancellous tissue and, under conditions of musculoskeletal disuse, can enhance the sensitivity of bone cells, in particular osteoprogenitors, to the effects of radiation.


Assuntos
Elevação dos Membros Posteriores , Osteoblastos/citologia , Osteoblastos/efeitos da radiação , Osteoclastos/citologia , Osteoclastos/efeitos da radiação , Tíbia/citologia , Tíbia/efeitos da radiação , Irradiação Corporal Total , Animais , Diferenciação Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Íons Pesados , Isótopos de Ferro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doses de Radiação
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