Pluripotent Stem Cells Actively Regulate The Openness of their Heterochromatin


Packaging DNA into a small area like the nucleus of the cell does not occur unless that DNA is tightly wound into compact structures collectively known as chromatin. However, not all regions of the genome show the same degree of compaction. Highly-expressed regions of the genome tend to be less highly compacted and regions of the genes that are not expressed to any degree tend to be squirreled away into tight chromatin.

Pluripotent stem cells tend to have an open and decondensed chromatin organization. In fact, this open and decondensed chromatin configuration is a defining property of pluripotent cells in general. The connection between pluripotency and the is open chromatin organization and the mediators of this chromatin configuration remain shrouded in uncertainty.

A new study from the laboratory of Peter J Rugg-Gunn at the Babraham Institute, in collaboration with scientists from Canada, the United Kingdom, and Japan, has identified two proteins, Nanog and Sall1 that participate in the chromatin structure of pluripotent stem cells. Such an understanding can contribute to making better pluripotent stem cells.

Cells tend to possess regions of the genome that are tightly wrapped into tight heterochomatin. These genomic regions are usually structural in nature and are, typically, not expressed. These include centromeric DNA and pericentromeric DNA, which plays a role in spindle attachment during cell division. These regions are collectively known as “constitutive heterochromatin.” However, previous research has demonstrated that this constitutive heterchromatin is maintained in an open and uncompacted conformation.

Clara Lopes Novo, in Rugg-Gunn’s laboratory and her colleagues discovered that transcription factor NANOG acts as an integral regulator of the conformation of constitutive heterochromatin in mouse embryonic stem cells. When Lopes Novo and others deleted the Nanog genes in mouse embryonic stem cells, the constitutive heterochromatin was remodeled in a manner that led to more intensive chromatin compaction. However, when Lopes Novo and her coworkers forced the expression of the Nanog gene in mouse embryonic stem cells, leading to spikes in the levels of NANOG proteim, the heterochromatin domains showed distinct decompaction.

When Lopes Novo and others determined where NANOG spent its time, they discovered that it was bound to heterochromatin. In particular, NANOG associated with satellite repeats within heterochromatin domains. Heterochromatin that was associated with NANOG had highly dispersed chromatin fibers, low levels of modified histone proteins that are usually associated with chromatin compaction (i.e. H3K9me3), and high levels of transcription.

The second heterochromatin-associated protein, SALL1, seems to work in cahoots with NANOG. In fact, when Lopes Novo and others deleted the Sall1 gene from mouse embryonic stem cells, the Sall1-/- cells recapitulate the Nanog -/- phenotype. However, further work showed that the loss of Sall1 can be rescued by forcing the recruitment of the NANOG to major portions of the heterochromatin (by over-expressing the NANOG protein).

These results demonstrate the connection between pluripotency and chromatin organization. This work seems to say, “embryonic stem cells actively maintain an open heterochromatin architecture.” They do this to stabilize their pluritotency.

Loss of heterochromatin regulation has potential consequences for the long-term genetic stability of stem cells, and the ability of stem cells, and the ability of stem cells to differentiate and mature into specialized cell types.

This work was published in the journal Genes and Development (http://www.genesdev.org/cgi/doi/10.1101/gad.275685.115)

Kidney Tubular Cells Formed from Stem Cells


A collaborative effort between several research teams has successfully directed stem cells to differentiate into kidney tubular cells. This is a significant advance that could hasten the day when stem cell-based treatments are used to treat kidney failure.

Chronic kidney disease is a major global public health problem. Unfortunately, once patients progress to kidney failure, their treatment options are limited to dialysis and kidney transplantation. Regenerative medicine, whose goal is to rebuild or repair tissues and organs, might offer a promising alternative.

A team of researchers from the Harvard Stem Cell Institute (Cambridge, Mass.), Brigham and Women’s Hospital (Boston) and Keio University School of Medicine (Tokyo) that included Albert Lam, M.D., Benjamin Freedman, Ph.D. and Ryuji Morizane, M.D., Ph.D., has been diligently developing strategies for the past five years to develop strategies to direct human pluripotent stem cells (human embryonic stem cells or hESCs and human induced pluripotent stem cells or iPSCs) to differentiate into kidney cells for the purposes of kidney regeneration.

“Our goal was to develop a simple, efficient and reproducible method of differentiating human pluripotent stem cells into cells of the intermediate mesoderm, the earliest tissue in the developing embryo that is fated to give rise to the kidneys,” said Dr. Lam. Lam also noted that these intermediate mesoderm cells would be the “starting blocks” for deriving more specific kidney cells.

Lam and his collaborators discovered a blend of chemicals which, when added to stem cells in a precise sequence, caused the stem cells to turn off their stem cell-specific genes and activate those genes found in kidney cells. Furthermore, the activation of the kidney-specific genes occurred in the same order that they turn on during embryonic kidney development.

At E10.5, the metanephric mesenchyme (red) comprises a unique subpopulation of the nephrogenic cord (yellow). Expression of the Glial-derived neurotrophic factor (Gdnf) is resticted to the metanephric mesenchyme by the actions of transcriptional activators, secreted factors, and inhibitors. GDNF binds the Ret receptor and promotes the formation of the ureteric bud, an outgrowth from the nephric duct (blue). Ret initially depends upon the Gata3 transcription factor for its expression in the nephric duct. Spry1 acts as an intracellular inhibitor of the Ret signal transduction pathway. BMP4 inhibits GDNF signaling and is in turn inhibited by the Grem1 binding protein. At 11.5, the ureteric bud has branched, forming a T-shaped structure. Each ureteric bud tip is surrounded by a cap of condensed metanephric mesenchyme. Reciprocal signaling between the cap mesenchyme and ureteric bud, as well as signals coming from stromal cells (red), maintain expression of Ret in the bud tips and Gdnf in the cap mesenchyme. Nephrons are derived from cap mesenchyme cells that form pretubular aggregates and then renal vesicles on either side of each ureteric bud tip. Wnt9b and Wnt4 induce nephron formation and are necessary for maintaining ureteric bud branching. The Six2 transcription factor prevents ectopic nephron formation. BMP7 promotes survival of the cap mesenchyme. Not all genes implicated in metanephros formation are shown for clarity (see text for further details). Green arrows indicate the ligand-receptor interaction between GDNF and Ret. Black arrows indicate the epistasis between genes but in most cases it is not known if the interactions are direct. T-shaped symbols indicate inhibitory interactions.
At E10.5, the metanephric mesenchyme (red) comprises a unique subpopulation of the nephrogenic cord (yellow). Expression of the Glial-derived neurotrophic factor (Gdnf) is resticted to the metanephric mesenchyme by the actions of transcriptional activators, secreted factors, and inhibitors. GDNF binds the Ret receptor and promotes the formation of the ureteric bud, an outgrowth from the nephric duct (blue). Ret initially depends upon the Gata3 transcription factor for its expression in the nephric duct. Spry1 acts as an intracellular inhibitor of the Ret signal transduction pathway. BMP4 inhibits GDNF signaling and is in turn inhibited by the Grem1 binding protein. At 11.5, the ureteric bud has branched, forming a T-shaped structure. Each ureteric bud tip is surrounded by a cap of condensed metanephric mesenchyme. Reciprocal signaling between the cap mesenchyme and ureteric bud, as well as signals coming from stromal cells (red), maintain expression of Ret in the bud tips and Gdnf in the cap mesenchyme. Nephrons are derived from cap mesenchyme cells that form pretubular aggregates and then renal vesicles on either side of each ureteric bud tip. Wnt9b and Wnt4 induce nephron formation and are necessary for maintaining ureteric bud branching. The Six2 transcription factor prevents ectopic nephron formation. BMP7 promotes survival of the cap mesenchyme. Not all genes implicated in metanephros formation are shown for clarity (see text for further details). Green arrows indicate the ligand-receptor interaction between GDNF and Ret. Black arrows indicate the epistasis between genes but in most cases it is not known if the interactions are direct. T-shaped symbols indicate inhibitory interactions.

The investigators were able to differentiate both hESCs and human iPSCs into cells that expressed the PAX2 and LHX1 genes, which are two key elements of the intermediate mesoderm; the developmental tissue from which the kidney develops. The iPSCs were derived by reprogramming fibroblasts obtained from adult skin biopsies into pluripotent cells. The differentiated cells expressed multiple genes found in intermediate mesoderm and spontaneously produced tubular structures that expressed those genes found in mature kidney tubules.

The researchers could then differentiate the intermediate mesoderm cells into kidney precursor cells that expressed the SIX2, SALL1 and WT1 genes. These three genes designate an embryonic tissue called the “metanephric cap mesenchyme.” Metanephric cap mesenchyme is a critical tissue for kidney differentiation. During kidney development, the metanephric cap mesenchyme contains a population of progenitor cells that give rise to nearly all of the epithelial cells of the kidney (epithelial cells or cells in a sheet, generate the lion’s share of the tubules of the kidney).

Metanephric cap mesenchyme is is red
Metanephric cap mesenchyme is is red

The cells also continued to behave like kidney cells when transplanted into adult or embryonic mouse kidneys. This gives further hope that these investigators might one day be able to create kidney tissues that could function in a patient and would be fully compatible with the patient’s immune system.

The findings are published online in Journal of the American Society of Nephrology.