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Anguera Laboratory Image Gallery


Thanks to advances in imaging techniques, you can view Dr. Anguera's research at both the cellular and sub-cellular levels. Get a virtual tour of some of the exciting work taking place in the Anguera Lab at Penn Vet.
Pachytene spermatocyte with synapsed chromosomes

Pachytene spermatocyte with synapsed chromosomes

This is the nucleus of a pachytene spermatocyte. We used immunofluorescence to detect two proteins: the histone variant H2AX (red) and SCP1 (green). The autosomes are paired together in this cell, and the SCP1 protein localizes along the entire synapsed chromosomes. The sex chromosomes (XY) lack pairing partners, and associate together. The H2AX protein localizes to unpaired chromosomes in pachytene spermatocytes, identifying the X and Y-chromosomes.

Cell Culture in the Anguera Lab

Cell Culture in the Anguera Lab

We culture a variety of mammalian cell lines: human and mouse pluripotent stem cells, human transformed and cancer cell lines. We culture our pluripotent stem cell lines at low oxygen levels, which reduces cellular differentiation.

Female Mouse Embryonic Stem Cells

Female Mouse Embryonic Stem Cells

These are mutant female mouse embryonic stem cells that were isolated from day 3.5 blastocysts. They are co-cultured with mouse embryonic feeder cells, which produce growth factors necessary to keep the embryonic stem cells undifferentiated. These embryonic stem cells have been manipulated to delete the X-linked long noncoding RNA Tsx.

Human Ovarian Cancer Cells lack XIST RNA

Human Ovarian Cancer Cells lack XIST RNA

These female cancer cells do not express XIST RNA. We used RNA fluorescence in situ hybridization (FISH) analyses with a probe for the human long noncoding RNA XIST (red) and a probe for Cot1 RNA (green). These cells have irreversibly silenced the XIST gene. Loss of XIST RNA expression is often observed in female cancer cell lines.

Culturing human pluripotent stem cells

Culturing human pluripotent stem cells

We use human pluripotent stem cells to investigate how long noncoding RNAs regulate gene expression during early human development.

Mouse Preimplantation Blastocyst

Mouse Preimplantation Blastocyst

These are two mouse blastocysts at day 3.5 of development. These embryos exhibit physical separation of the inner cell mass (which forms the embryo) from the trophectoderm (which forms the placenta). This is the first cell lineage specification event during mammalian development. These embryos were used to derive mouse embryonic stem cell lines, generated by culturing the inner cell mass cells on a layer of embryonic feeder cells.

Apoptotic germ cells in mice

Apoptotic germ cells in mice

This is a testes section from a mutant mouse (missing the X-linked noncoding RNA Tsx). We performed a TUNEL assay to identify the germ cells undergoing cell death. The green cells are apopototic spermatocytes (nuclei are stained with DAPI).

 Transcriptome analysis of human pluripotent stem cells

Transcriptome analysis of human pluripotent stem cells

We are generating catalogues of female-specific long noncoding RNAs expressed in high and low quality human pluripotent stem cells. These are strand-specific RNA-Sequencing results for a particular region of the human genome for a novel long noncoding RNA. It contains the characteristic chromatin marks of active transcription: H3K4me modifications at the promoter, and H3K36me3 along the gene body.

Human female induced pluripotent stem cells

Human female induced pluripotent stem cells

This is a picture of a colony of human pluripotent stem cells. We used the Yamanaka 4-Factors to reprogram female fibroblasts into ‘induced pluripotent stem cells’ (iPSCs). These hiPSCs are grown together with mouse embryonic feeder cells.

The long noncoding RNA XIST coats the silent X-chromosome

The long noncoding RNA XIST coats the silent X-chromosome

This is a nucleus of a human female induced pluripotent stem cell (reprogrammed from a fibroblast cell). We used fluorescence in situ hybridization (FISH) to detect the long noncoding RNA XIST (red), nascent transcription using a probe for Cot1 DNA (green), and DAPI to visualize nuclei (blue). The reprogramming process does not reactive the inactive X-chromosome in human somatic cells (unlike mouse cells).

Newly reprogrammed human induced pluripotent stem cells express pluripotency markers

Newly reprogrammed human induced pluripotent stem cells express pluripotency markers

This is a colony of human female induced pluripotent stem cells that we reprogrammed in our lab. We used immuno-fluorescence to detect expression of the transcription factor OCT4 (POU5F1), which is required for regulating pluripotency during normal development. OCT4 is one of the four “Yamanaka” factors used to reprogram somatic cells to the pluripotent state.

Heterochromatin of the human inactive X-chromosome

Heterochromatin of the human inactive X-chromosome

This is a nucleus of a human female induced pluripotent stem cell (reprogrammed from a fibroblast cell). We used immunofluorescence to detect the H3K27me3 modification (red), a histone modification enriched on the inactive X-chromosome in female somatic cells. The nucleus is stained with DAPI (blue). The reprogramming process does not reactive the inactive X-chromosome in human somatic cells, so the H3K27me3 focus of the inactive X is retained (unlike mouse cells).

A colony of human induced pluripotent stem cells

A colony of human induced pluripotent stem cells

This is a colony of human female induced pluripotent stem cells (hiPSCs) that have stopped expressing the long noncoding RNA XIST from one of the X-chromosomes. These cells look identical to other female hiPSCs that express XIST RNA. We have found that XIST-expressing and XIST-silent cell lines are quite different at the molecular level: over 300 genes are differentially expressed, and there’s partial re-activation of the inactive X-chromosome.

Newly reprogrammed human induced pluripotent stem cells express pluripotency markers

Newly reprogrammed human induced pluripotent stem cells express pluripotency markers

This is a colony of human female induced pluripotent stem cells that we reprogrammed in our lab. We used immuno-fluorescence to detect expression of the cell surface marker Tra-1-60, which is only expressed in pluripotent stem cells.

Female mouse embryonic stem cells express the long noncoding RNA Xist from both X-chromosomes

Female mouse embryonic stem cells express the long noncoding RNA Xist from both X-chromosomes

Female mouse embryonic stem cells (shown here) and the cells of the inner cell mass of preimplantation embryos have two active X-chromosomes. These cells express low levels (about 4 copies/cell) of the long noncoding RNA Xist, which can be detected using RNA fluorescence in situ hybridization. Upon differentiation, one X is chosen for transcriptional silencing and upregulates Xist RNA expression.

In vitro differentiation of mouse embryonic stem cells into embryoid bodies

In vitro differentiation of mouse embryonic stem cells into embryoid bodies

Pluripotent stem cells can be differentiated in vitro to form the three germ lineages. Here we differentiated female mouse embryonic stem cells to form embryoid bodies (EBs), which are three-dimensional aggregates of stem cells cultured in medium lacking factors that support pluripotency (without LIF). These EBs have been differentiating for four days.

Karyotype analysis of human female induced pluripotent stem cells

Karyotype analysis of human female induced pluripotent stem cells

We use karyotype analysis to determine whether cellular reprogramming has altered the number of chromosomes. Karyotype analysis can detect large chromosomal abnormalities (loss or gain of an entire chromosome or portions of a chromosome) or translocations (when a portion of a chromosome breaks off and rejoins with another chromosome). These are the results from one human female induced pluripotent stem cell line at passage 30, which remained karyotypically normal after cellular reprogramming and routine culture.

Comparative genomic hybridization analysis of human pluripotent stem cells

Comparative genomic hybridization analysis of human pluripotent stem cells

Comparative genomic hybridization is a cytogenetic method to determine copy number variation in genomic DNA. Here we compared human female pluripotent stem cell lines that express XIST RNA to parental lines that have silenced the XIST gene. These samples are nearly identical, indicating that the gene expression differences in cell lines lacking XIST expression are not due to chromosomal abnormalities.

Genome editing of human pluripotent stem cells

Genome editing of human pluripotent stem cells

These are human pluripotent stem cells that have been engineered to express GFP from the AAVS.1 locus on chromosome 19. We used a set of TALE nucleases to introduce double-strand breaks in the intronic region of this gene, which facilitated homologous recombination of a GFP reporter gene (driven by the endogenous promoter).

Pachytene spermatocytes

Pachytene spermatocytes

These are mouse germ cells that are undergoing meiosis. At this stage, the chromosome pairs are aligned together. We used immunofluorescence to detect the SCP1 protein, which localizes along the entire length of each synapsed chromosome.

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  • Pachytene spermatocyte with synapsed chromosomes
  • Cell Culture in the Anguera Lab
  • Female Mouse Embryonic Stem Cells
  • Human Ovarian Cancer Cells lack XIST RNA
  • Culturing human pluripotent stem cells
  • Mouse Preimplantation Blastocyst
  • Apoptotic germ cells in mice
  •  Transcriptome analysis of human pluripotent stem cells
  • Human female induced pluripotent stem cells
  • The long noncoding RNA XIST coats the silent X-chromosome
  • Newly reprogrammed human induced pluripotent stem cells express pluripotency markers
  • Heterochromatin of the human inactive X-chromosome
  • A colony of human induced pluripotent stem cells
  • Newly reprogrammed human induced pluripotent stem cells express pluripotency markers
  • Female mouse embryonic stem cells express the long noncoding RNA Xist from both X-chromosomes
  • In vitro differentiation of mouse embryonic stem cells into embryoid bodies
  • Karyotype analysis of human female induced pluripotent stem cells
  • Comparative genomic hybridization analysis of human pluripotent stem cells
  • Genome editing of human pluripotent stem cells
  • Pachytene spermatocytes