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.

Scientists Generate “Mini-kidney” Structures from Human Stem Cells


Kidney Disease represents a major and unsolved health issue worldwide. Once damaged by disease, kidneys rarely recover their original level of function, and this highlights the urgent need for better knowledge of kidney development and physiology.

Now, a team of researchers led by scientists at the Salk Institute for Biological Studies has developed a novel platform to study kidney diseases. This new platform should open new avenues for the future application of regenerative medical strategies to restore kidney function.

For the first time, the Salk researchers have generated three-dimensional kidney structures from human stem cells. These findings were reported November 17, 2013 in Nature Cell Biology, and they suggest new ways to study the development and diseases of the kidneys and to discover and test new drugs that target human kidney cells.

Scientists had created precursors of kidney cells using stem cells as recently as this past summer, but the Salk team was the first to coax human stem cells into forming three-dimensional cellular structures similar to those found in our kidneys.

“Attempts to differentiate human stem cells into renal cells have had limited success,” says senior study author Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and holder of the Roger Guillemin Chair. “We have developed a simple and efficient method that allows for the differentiation of human stem cells into well-organized 3D structures of the ureteric bud (UB), which later develops into the collecting duct system.”

The Salk findings demonstrate for the first time that pluripotent stem cells capable of differentiating into the many cells and tissue types that make up the body can be induced to differentiate into those cells found in the ureteric bud, which is an early developmental structure of the kidneys. Furthermore, these same cells can differentiate further into three-dimensional structures in organ cultures. Ureteric bud cells form the early stages of the human urinary and reproductive organs during development and later develop into a conduit for urine drainage from the kidneys. Izpisua Belmonte’s research group accomplished this with both human embryonic stem cells and induced pluripotent stem cells (iPSCs), human cells from the skin that have been reprogrammed into their pluripotent state.

Kidney development

After generating iPSCs that demonstrated pluripotent properties and were able to differentiate into mesoderm, the embryonic germ cell layer from which the kidneys develop, the Salk Institute team used growth factors known to be essential during the natural development of our kidneys to culture both iPSCs and embryonic stem cells.  The combination of signals from these growth factors, molecules that guide the differentiation of stem cells into specific tissues, committed the cells to become progenitors that exhibit clear characteristics of renal cells in only four days.

The researchers then guided these cells to further differentiate into organ structures similar to those found in the ureteric bud by culturing them with kidney cells from mice. This demonstrated that the mouse cells were able to provide the appropriate developmental cues to allow human stem cells to form three-dimensional structures of the kidney.

Izpisua Belmonte’s team also tested their protocol on iPSCs from a patient clinically diagnosed with polycystic kidney disease (PKD), a genetic disorder characterized by multiple, fluid-filled cysts that can lead to decreased kidney function and kidney failure. They found that their methodology could produce kidney structures from patient-derived iPSCs.

Polycystic kidneys
Polycystic kidneys

Because of the many clinical manifestations of the disease, neither gene- nor antibody-based therapies are realistic approaches for treating PKD. The Salk team’s technique might help circumvent this obstacle and provide a reliable platform for pharmaceutical companies and other investigators studying drug-based therapeutics for PKD and other kidney diseases.

“Our differentiation strategies represent the cornerstone of disease modeling and drug discovery studies,” says lead study author Ignacio Sancho-Martinez, a research associate in Izpisua Belmonte’s laboratory. “Our observations will help guide future studies on the precise cellular implications that PKD might play in the context of kidney development.”