RESUMEN
Tuberculosis (TB) in humans is characterized by formation of immune-rich granulomas in infected tissues, the architecture and composition of which are thought to affect disease outcome. However, our understanding of the spatial relationships that control human granulomas is limited. Here, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) to image 37 proteins in tissues from patients with active TB. We constructed a comprehensive atlas that maps 19 cell subsets across 8 spatial microenvironments. This atlas shows an IFN-γ-depleted microenvironment enriched for TGF-ß, regulatory T cells and IDO1+ PD-L1+ myeloid cells. In a further transcriptomic meta-analysis of peripheral blood from patients with TB, immunoregulatory trends mirror those identified by granuloma imaging. Notably, PD-L1 expression is associated with progression to active TB and treatment response. These data indicate that in TB granulomas, there are local spatially coordinated immunoregulatory programs with systemic manifestations that define active TB.
Asunto(s)
Granuloma/inmunología , Tuberculosis/inmunología , Antígeno B7-H1/inmunología , Células Cultivadas , Citocinas/inmunología , Perfilación de la Expresión Génica/métodos , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Pulmón/inmunología , Mycobacterium tuberculosis/inmunología , Células Mieloides/inmunologíaRESUMEN
The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.
Asunto(s)
Linfocitos/inmunología , Espectrometría de Masas , Neoplasias de la Mama Triple Negativas/patología , Microambiente Tumoral/inmunología , Antígenos CD/metabolismo , Antígeno B7-H1/metabolismo , Análisis por Conglomerados , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Estimación de Kaplan-Meier , Linfocitos/citología , Linfocitos/metabolismo , Aprendizaje Automático , Análisis de Componente Principal , Receptor de Muerte Celular Programada 1/metabolismo , Análisis Espacial , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/mortalidad , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
Asunto(s)
Infecciones por VIH , Ácidos Nucleicos , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD4-Positivos , Virus ADN , Terapia de Inmunosupresión , Macaca mulatta , Macrófagos , Virus de la Inmunodeficiencia de los Simios/fisiología , Carga ViralRESUMEN
Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.
Asunto(s)
Intercambio Materno-Fetal , Trofoblastos , Útero , Femenino , Humanos , Embarazo , Arterias/fisiología , Decidua/irrigación sanguínea , Decidua/citología , Decidua/inmunología , Decidua/fisiología , Primer Trimestre del Embarazo/genética , Primer Trimestre del Embarazo/metabolismo , Primer Trimestre del Embarazo/fisiología , Trofoblastos/citología , Trofoblastos/inmunología , Trofoblastos/fisiología , Útero/irrigación sanguínea , Útero/citología , Útero/inmunología , Útero/fisiología , Intercambio Materno-Fetal/genética , Intercambio Materno-Fetal/inmunología , Intercambio Materno-Fetal/fisiología , Factores de Tiempo , Proteómica , Perfilación de la Expresión Génica , Conjuntos de Datos como Asunto , Edad GestacionalRESUMEN
Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) is a form of mass spectrometry imaging that uses metal labeled antibodies and secondary ion mass spectrometry to image dozens of proteins simultaneously in the same tissue section. Working with the National Cancer Institute's (NCI) Cancer Immune Monitoring and Analysis Centers (CIMAC), we undertook a validation study, assessing concordance across a dozen serial sections of a tissue microarray of 21 samples that were independently processed and imaged by MIBI-TOF or single-plex immunohistochemistry (IHC) over 12 days. Pixel-level features were highly concordant across all 16 targets assessed in both staining intensity (R2 = 0.94 ± 0.04) and frequency (R2 = 0.95 ± 0.04). Comparison to digitized, single-plex IHC on adjacent serial sections revealed similar concordance (R2 = 0.85 ± 0.08) as well. Lastly, automated segmentation and clustering of eight cell populations found that cell frequencies between serial sections yielded an average correlation of R2 = 0.94 ± 0.05. Taken together, we demonstrate that MIBI-TOF, with well-vetted reagents and automated analysis, can generate consistent and quantitative annotations of clinically relevant cell states in archival human tissue, and more broadly, present a scalable framework for benchmarking multiplexed IHC approaches.
Asunto(s)
Diagnóstico por Imagen , Neoplasias , Anticuerpos , Diagnóstico por Imagen/métodos , Humanos , Inmunohistoquímica , Iones , Espectrometría de Masas/métodosRESUMEN
Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-µm thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging â¼42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.
Asunto(s)
Proteínas , Análisis de la Célula Individual , Immunoblotting , Iones , Espectrometría de MasasRESUMEN
Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction.
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Adenosina Trifosfatasas/química , Carboxipeptidasas/química , Regulación Enzimológica de la Expresión Génica , Complejo de la Endopetidasa Proteasomal/química , Ubiquitina Tiolesterasa/química , Ubiquitina/química , Adenosina Trifosfato/química , Animales , Fibroblastos/metabolismo , Proteínas Fúngicas , Hidrólisis , Ratones , Modelos Biológicos , Conformación Molecular , Estructura Terciaria de Proteína , ConejosRESUMEN
Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Células Madre Pluripotentes/citología , Somatomedinas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Proliferación Celular , Medios de Cultivo Condicionados/química , Factores de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Humanos , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Proteoma/metabolismo , Receptor IGF Tipo 1/deficiencia , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Somatomedinas/biosíntesis , Somatomedinas/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.
Asunto(s)
Virus del Dengue , Dengue , Dengue Grave , Niño , Humanos , Cinética , Proteómica , Inmunidad InnataRESUMEN
Microglia are implicated in aging, neurodegeneration, and Alzheimer's disease (AD). Traditional, low-plex, imaging methods fall short of capturing in situ cellular states and interactions in the human brain. We utilized Multiplexed Ion Beam Imaging (MIBI) and data-driven analysis to spatially map proteomic cellular states and niches in healthy human brain, identifying a spectrum of microglial profiles, called the microglial state continuum (MSC). The MSC ranged from senescent-like to active proteomic states that were skewed across large brain regions and compartmentalized locally according to their immediate microenvironment. While more active microglial states were proximal to amyloid plaques, globally, microglia significantly shifted towards a, presumably, dysfunctional low MSC in the AD hippocampus, as confirmed in an independent cohort (n=26). This provides an in situ single cell framework for mapping human microglial states along a continuous, shifting existence that is differentially enriched between healthy brain regions and disease, reinforcing differential microglial functions overall.
RESUMEN
Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.
Asunto(s)
Microscopía , Proteínas , Humanos , Vacio , Microscopía/métodos , Hidrogeles/químicaRESUMEN
Next-generation tools for multiplexed imaging have driven a new wave of innovation in understanding how single-cell function and tissue structure are interrelated. In previous work, we developed multiplexed ion beam imaging by time of flight, a highly multiplexed platform that uses secondary ion mass spectrometry to image dozens of antibodies tagged with metal reporters. As instrument throughput has increased, the breadth and depth of imaging data have increased as well. To extract meaningful information from these data, we have developed tools for cell identification, cell classification, and spatial analysis. In this review, we discuss these tools and provide examples of their application in various contexts, including ductal carcinoma in situ, tuberculosis, and Alzheimer's disease. We hope the synergy between multiplexed imaging and automated image analysis will drive a new era in anatomic pathology and personalized medicine wherein quantitative spatial signatures are used routinely for more accurate diagnosis, prognosis, and therapeutic selection.
Asunto(s)
Inmunohistoquímica , Espectrometría de Masas , Anticuerpos , Humanos , Inmunohistoquímica/métodos , Espectrometría de Masas/métodosRESUMEN
Neurodegenerative disorders are characterized by phenotypic changes and hallmark proteopathies. Quantifying these in archival human brain tissues remains indispensable for validating animal models and understanding disease mechanisms. We present a framework for nanometer-scale, spatial proteomics with multiplex ion beam imaging (MIBI) for capturing neuropathological features. MIBI facilitated simultaneous, quantitative imaging of 36 proteins on archival human hippocampus from individuals spanning cognitively normal to dementia. Customized analysis strategies identified cell types and proteopathies in the hippocampus across stages of Alzheimer's disease (AD) neuropathologic change. We show microglia-pathologic tau interactions in hippocampal CA1 subfield in AD dementia. Data driven, sample independent creation of spatial proteomic regions identified persistent neurons in pathologic tau neighborhoods expressing mitochondrial protein MFN2, regardless of cognitive status, suggesting a survival advantage. Our study revealed unique insights from multiplexed imaging and data-driven approaches for neuropathologic analysis and serves broadly as a methodology for spatial proteomic analysis of archival human neuropathology. TEASER: Multiplex Ion beam Imaging enables deep spatial phenotyping of human neuropathology-associated cellular and disease features.
Asunto(s)
Enfermedad de Alzheimer , Proteómica , Animales , Humanos , Neuropatología , Enfermedad de Alzheimer/patología , Hipocampo/patología , Microglía/patología , Proteínas tau/metabolismoRESUMEN
The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45(+)CD34(+) cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45(+)CD34(-)) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.
Asunto(s)
Células Madre Embrionarias/citología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células del Estroma/citología , Animales , Animales Recién Nacidos , Antígenos CD34/metabolismo , Apoptosis , Línea Celular , Proliferación Celular , Separación Celular , Supervivencia Celular , Técnicas de Cocultivo , Proteínas de Homeodominio/metabolismo , Humanos , Antígenos Comunes de Leucocito/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
Understanding tissue structure and function requires tools that quantify the expression of multiple proteins while preserving spatial information. Here, we describe MIBI-TOF (multiplexed ion beam imaging by time of flight), an instrument that uses bright ion sources and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies at subcellular resolution in clinical tissue sections. We demonstrate quantitative, full periodic table coverage across a five-log dynamic range, imaging 36 labeled antibodies simultaneously with histochemical stains and endogenous elements. We image fields of view up to 800 µm × 800 µm at resolutions down to 260 nm with sensitivities approaching single-molecule detection. We leverage these properties to interrogate intrapatient heterogeneity in tumor organization in triple-negative breast cancer, revealing regional variability in tumor cell phenotypes in contrast to a structured immune response. Given its versatility and sample back-compatibility, MIBI-TOF is positioned to leverage existing annotated, archival tissue cohorts to explore emerging questions in cancer, immunology, and neurobiology.
Asunto(s)
Espectrometría de Masa de Ion Secundario/métodos , Anticuerpos/química , Anticuerpos/inmunología , Astrocitos/citología , Astrocitos/metabolismo , Proteína Ácida Fibrilar de la Glía/química , Proteína Ácida Fibrilar de la Glía/inmunología , Humanos , Metales/química , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/inmunología , Microglía/citología , Microglía/metabolismo , Tonsila Palatina/patología , Fenotipo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
During human development, signals that govern lineage specification versus expansion of cells committed to a cell fate are poorly understood. We demonstrate that activation of canonical Wnt signaling by Wnt3a promotes proliferation of human embryonic stem cells (hESCs)--precursors already committed to the hematopoietic lineage. In contrast, noncanonical Wnt signals, activated by Wnt11, control exit from the pluripotent state and entry toward mesoderm specification. Unique to embryoid body (EB) formation of hESCs, Wnt11 induces development and arrangement of cells expressing Brachyury that coexpress E-cadherin and Frizzled-7 (Fzd7). Knockdown of Fzd7 expression blocks Wnt11-dependent specification. Our study reveals an unappreciated role for noncanonical Wnt signaling in hESC specification that involves development of unique mesoderm precursors via morphogenic organization within human EBs.
Asunto(s)
Células Madre Embrionarias/fisiología , Hematopoyesis/fisiología , Proteínas Wnt/metabolismo , Tipificación del Cuerpo , Cadherinas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Madre Embrionarias/efectos de los fármacos , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Hematopoyesis/efectos de los fármacos , Humanos , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Wnt/farmacología , Proteína Wnt3 , Proteína Wnt3ARESUMEN
Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal, teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced 'stemness'. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor independence, a 9- to 20-fold increase in frequency of tumor-initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization reveals an amplification at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional characterization to distinguish partially transformed and normal hES cells.
Asunto(s)
Células Madre Embrionarias/citología , Neoplasias/patología , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Aberraciones Cromosómicas , Hibridación Genómica Comparativa , Citogenética , Progresión de la Enfermedad , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Técnicas Genéticas , Humanos , Hibridación de Ácido Nucleico , Fenotipo , Células Madre/metabolismoRESUMEN
Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3-positive (SSEA-3+) and SSEA-3- cells from hESC cultures. Although both SSEA-3+ and SSEA-3- cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.