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.

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Cloned embryos can’t fool a womb


See the following link for an interesting paper on the ability of the womb to discern between a cloned embryo and an embryo made by means of fertilization. See this link for the article.

Embryonic stem cells from cloned embryos are indistinguishable from those made from embryos made by fertilization.  They express the same genes (see  DJ Guo et al., Proteomics. 2009 Apr 22, and this article), show the same biological behaviors (see this link for this paper), show normal embryonic stem cell morphology, express key stem-cell markers, and can differentiate into multiple cell types in vitro and in vivo (JA Byrne, Nature 450 (2007): 497-502).

Since embryonic stem cells are made from the internal cells of the embryo (the inner cell mass), the inner cell mass cells from cloned embryos are rather normal (ML Condic, Cell Proliferation 41, suppl 1: 7-19).  However, the outer layer of cells (trophectoderm) that engage the endometrium and work with it to implant the embryo into the inner layer of the uterus do not differentiate normally in cloned embryos (DR Arnold et al. Reproduction 132, no. 2 (2006): 279­-90).  Trophoblast cells  in cloned embryos are normal at the early stages (S. Kishigami et al., FEBS Letters 580, no. 7 (2006): 1801-6), but they go on to make abnormal placentas (DR Arnold et al, Placenta 29 Suppl A (2007): S108-10).

Now this paper shows that the differentiating placenta of the cloned embryo does not interact normally with the surrogate mother’s uterus.  This is probably one of the main reasons why cloned embryos and fetuses tend to die prior to birth.  The endometrial cells of the mothers who were carried the cloned embryos showed substantial variation in the genes they expressed in comparison to endometria that carried in vitro fertilized embryos (S. Bauersachs et al., PNAS 106, no. 14 (2009): 5681-6).  Thus cloned embryos fail to properly communicate with the mother’s uterus.

Implantation is a very complex process.  It requires cross talk between the embryo and the uterus.  Without this cross talk, implantation does not occur successfully.  Without successful implantation, the embryo perishes.

Here again we find another reason to not clone humans.  We are subjecting them to a process that is less robust than fertilization.  The chances of the embryo surviving are far less than an embryo concieved in the usual manner (fertilization).  We should simply ban this process in humans overall.