New 3D Method Used to Grow Miniature Pancreas


Researchers from the University of Copenhagen, in collaboration with an international team of investigators, have successfully developed an innovative three-dimensional method to grow miniature pancreas from progenitor cells. The future goal of this research is to utilize this model system to fight against diabetes. This research was recently published in the journal Development.

The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.
The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.

The new method takes cell material from mice and grows them in vividly picturesque tree-like structures.  The cells used were mouse embryonic pancreatic progenitors, and they were grown in a compound called Matrigel with accompanying cocktails of growth factors.  In vitro maintenance and expansion of these pancreatic progenitors requires active Notch and FGF signaling, and therefore, this culture system recapitulated the in vivo conditions that give rise to the pancreas in the embryo.

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre, in collaboration with colleagues from the Ecole Polytechnique Fédérale de Lausanne in Switzerland, have developed a three-dimensional culture method that takes pancreatic cells and vigorously expands them. This new method allows the cell material from mice to grow vividly into several distinct picturesque, tree-like structures. The method offers tremendous long-term potential in producing miniature human pancreas from human stem cells. Human miniature pancreas organoids would be valuable as models to test new drugs fast and effectively, without the use of animal models.

“The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

In culture, pancreatic cells neither thrive nor develop if they are alone. A minimum of four pancreatic cells, growing close together is required for these cells to undergo organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment, and are influenced by such seemingly insignificant factors as the stiffness of the gel and contact with other cells.

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

Scientists Grow Small Chunks of Brain Tissue From Induced Pluripotent Stem Cells


Induced pluripotent stem cells are made from adult cells by means of genetic engineering techniques that introduce into the cells a combination for four different genes that drive the cells to de-differentiate into a cell that has many of the characteristics of embryonic stem cells without the destruction of embryos.

A new study from the laboratory of Juergen Knoblich at the Institute of Molecular Biotechnology in Vienna has mixed induced pluripotent stem cells (iPSCs) to form structures of the human brain. He largely left the cells alone to allow them to form the brain tissue, but he also placed them in a spinning bioreactor that constantly circulates the culture medium and provides nutrients and oxygen to the cells. One other growth factor he supplied to the cells was retinoic acid, which is made by the meninges that surround our brains. All of this and the cells not only divided, differentiated and assembled, but they formed brain structures that had all the connections of a normal brain. These brain-like chunks of tissue are called “mini-brains” and the recent edition of the journal Nature reports their creation.

“It’s a seminal study to making a brain in a dish,” says Clive Svendsen, a neurobiologist at the University of California, Los Angeles. Svendsen was not involved in this study, but wishes he was. Of this study, Svendsen exclaimed, “That’s phenomenal” A fully formed artificial brain is still years and years away, but the pea-sized neural clumps developed in Knoblich’s laboratory could prove useful for researching human neurological diseases.

Researchers have previously used pluripotent human stem cells to grow structures that resemble the developing eye (Eiraku, M. et al. Nature 472, 51–56 (2011), and even tissue layers similar to the cerebral cortex of the brain (Eiraku, M. et al. Cell Stem Cell 3, 519–532 (2008). However, this latest advance has seen bigger and more complex neural-tissue clumps by first growing the stem cells on a synthetic gel that resembled natural connective tissues found in the brain and elsewhere in the body. After growing them on the synthetic gel, Knoblich and his colleagues transferred the cells to a spinning bioreactor that infuses the cells with nutrients and oxygen.

“The big surprise was that it worked,” said Knoblich. The clump formed structures that resembled the brains of fetuses in the ninth week of development.

Under a microscope, the blobs contained discrete brain regions that seemed to interact with one another. However, the overall arrangement of the different proto-brain areas varied randomly across tissue samples. These structures were not recognizable physiological structures.

A cross-section of a brain-like clump of neural cells derived from human stem cells.
A cross-section of a brain-like clump of neural cells derived from human stem cells.

“The entire structure is not like one brain,” says Knoblich, who added that normal brain maturation in an intact embryo is probably guided by growth signals from other parts of the body. The tissue balls also lacked blood vessels, which could be one reason that their size was limited to 3–4 millimeters in diameter, even after growing for 10 months or more.

Despite these limitations, Knoblich and his collaborators used this system to model key aspects of microcephaly, which is a condition that causes extremely stunted brain growth and cognitive impairment. Microcephaly and other neurodevelopmental disorders are difficult to replicate in rodents because the brains of rodents develop differently than those of humans.

Knoblich and others found that tissue chunks cultured from stem cells derived from the skin of a single human with microcephaly did not grow as large as clumps grown from stem cells derived from a healthy person. When they traced this effect, they discovered that it was due to the premature differentiation of neural stem cells inside the microcephalic tissue chunks, which depleted the population of progenitor cells that fuels normal brain growth.

The findings largely confirm prevailing theories about microcephaly, says Arnold Kriegstein, a developmental neurobiologist at the University of California, San Francisco. But, he adds, the study also demonstrates the potential for using human-stem-cell-derived tissues to model other disorders, if cell growth can be controlled more reliably.

“This whole approach is really in its early stages,” says Kriegstein. “The jury may still be out in terms of how robust this is.”

Induced Pluripotent Stem Cells Replace Liver Function in Mice


Liver transplants save lives and in the United States there is a shortage of livers for transplantation. Between July 1, 2008 and June 30, 2011, well over 14,601 adult donor livers were recovered and transplanted. Of these livers that were transplanted, many other patients died from liver failure. If there was a way to restore liver function in patients with liver failure without dependence on a liver from a liver donor, then we might be able to extend their lives.

A paper from the laboratory of Hossein Baharvand at the University of Science and Culture in Tehran, Iran provides a step towards doing just that. In this paper, Baharvand and his colleagues used human induced pluripotent stem cells to make hepatocyte-like cells or HLCs. Hepatocyte is a fancy word for a liver cell. These HLCs were then transplanted into the spleen of mice that have damaged livers, and they rescued liver function in these mice.

The liver is a vital organ. It processes molecules absorbed by the digestive system, processes foreign chemicals to make them more easily excreted. It also produces bile, which helps dispose of fat-soluble waste and solubilize fats for degradation in the small intestine during digestion. It also produces blood plasma proteins, cholesterol and special proteins to cholesterol and fat transport, converts excess glucose into glycogen for storage, regulates blood levels of amino acids (the building blocks of proteins), processes used hemoglobin to recycle its iron content, converts poisonous ammonia to urea, regulates blood clotting, and helps the body resist infections by producing immune factors and removing bacteria from the bloodstream. Thus without a functioning liver, you are in deep weeds.

Induced pluripotent stem cells or iPSCs are made from adult cells that have been genetically engineered to de-differentiate into embryonic-like stem cells. They can be grown in culture to large numbers, and can also be differentiated into, potentially, any cell type in the adult body.

In this paper, Baharvand and his colleagues grew human iPSCs in “matrigel,” and then grew them in suspension. Matrigel is gooey and the cells stick to it and grow, and they were grown in matrigel culture for 1 week. After one week, the cells were grown in liquid suspension for 1-2 weeks. The cells have better access to soluble growth factors in liquid culture and tend to grow faster. After this they were grown in a stirred culture (known as a spinner).  This expanded the cells into large numbers for further use.

 Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.
Expansion and characterization of human induced pluripotent stem cells (hiPSCs) in a dynamic suspension culture. (A) Schematic representation of the suspension culture and expansion of human pluripotent stem cells (hPSCs) from adherent to stirred bioreactor. The cells were transferred to bacterial dishes to adapt to three-dimensional (3D) environment and then transferred into the dynamic phase, stirred flask. (B) Morphology of a tested hiPSC line (hiPSC1) after passage in a stirred suspension bioreactor as monitored by dark-field microscopy. (C) Immunostaining of cross sections of spheroids for OCT4 and TRA-1-81. Scale bar: 50 μm. (D) Flow cytometry analysis and (E) normal karyotype of suspended cells in the bioreactor.

Getting cells to grow in liquid suspension tends to be a bit of an art form, but these iPSCs grew rather well. Also, the iPSCs were differentiated into definitive endoderm, which is the first step in bringing cells to the liver cell stage. The drug Rapamycin and activin (50 ng / L for those who are interested) were used to bring the growing iPSCs to the definitive endoderm.  The cells expressed all kinds of endoderm-specific genes.  Endoderm is the embryonic germ layer from which the digestive system and its accessory organs forms.

 Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey's post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
Induction of hiPSCs into definitive endoderm. (A) Diagrammatic representation of the experimental groups for endoderm induction of hiPSCs in the bacterial dish static suspension, which include rapamycin (Rapa) “priming” and activin A “inducing” phases, and positive control groups of hiPSCs cultured in the absence of Rapa in suspension or adherent cultures in the presence of activin A. (B) Gene expression analysis of hiPSCs induced into endodermal cells. Quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) showed no significant differences in SOX17 and FOXA2 [definitive endoderm (DE) markers], and BRA (mesoendoderm marker) in all groups. SOX7 (visceral endoderm marker) was expressed at a low level. Influence of the refreshment strategy of the induction medium on DE formation in suspension cultures by qRT-PCR (C), immunostaining (D), and flow cytometry (E). We compared single (SR), double (DR), and triple (TR) refreshment of induction medium per 24 h for 4 days after Rapa administration in the static suspension of hiPSCs. Scale bar: 100 μm. The target gene expression level in qRT-PCR was normalized to GAPDH and calibrated with (presented relative to) hiPSCs. Data are presented as mean±SD. Statistical analysis as determined by one-way ANOVA with Tukey’s post hoc test, n=3 for B, C, and E. *P<0.05, **P<0.01.
After the cells went through this culture protocol, they were grown in a stirred liquid culture called a “spinner.” The culture system contain a cocktail of growth factors that differentiated the definitive endoderm cells into HLCs.  The cells formed little spheres that expressed a host of liver-specific genes.

Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.
Differentiation of hepatocyte-like cells (HLCs) from hiPSCs in the stirred bioreactor. (A) Stepwise protocol for differentiation of hiPSCs into HLCs. (B) The morphology and cross section of spheroids at day 21. The spheroids in this step were observed as cystic and dense spheroids. Hematoxylin and eosin (H&E) staining of spheroid cross sections indicated cystic and dense epithelioid appearances in the transplant and dense spheroids, respectively. Scale bar: 100 μm. (C) Comparative relative mRNA expression in cystic and dense spheroids normalized to GAPDH and calibrated to undifferentiated hiPSCs. Transplant and dense spheroids expressed more early and late hepatic lineage markers, respectively. Data are presented as mean. n=3. (D) Transmission electron microscopy (TEM) of differentiated cells in dense spheroids at day 21. Nucleus (N), nucleoli (n), mitochondria (M), Golgi apparatus (G), lysosomes (Ly), rough endoplasmic reticuli (arrowhead), glycogen granules (GR), intermediate filaments (CK), tight junctions (TJ), gap junctions (GJ), fascia adherens (FA), junctional complex (JC), microvilli (MV), and bile-like canaliculus (BLC). Scale bar: 1 μm.

From the figure above, we can see that these HLCs, not only express liver-specific genes, but when they are examined in the electron microscope they look, for all intents and purposes, like liver cells.  Functional tests of these spheres of HLCs showed that they 1) took up low-density lipoprotein; 2) produced albumin (a major blood plasma protein); 3) expressed cytochrome P450s, which are the major enzymes used to process drugs; 4) produced urea from amino acids, just like real liver cells; 5) accumulated glycogen; 6) and made liver proteins (HNF4a, ALB, etc).

So it looks like liver, quacks like liver, but can it replace liver?  These HLCs were transplanted into the spleen of mice whose livers had been treated with carbon tetrachloride.  Carbon tetrachloride tends to make mincemeat of the liver, and these mice are in trouble, since their livers are toast.  Transplantation of the iPSC-derived HLCs into the spleens of these mice increased their survival rate and decreased the blood levels of liver enzymes that are usually present when there is liver damage.

This paper is significant because the procedure used provides an example of a “scalable” protocol for making large quantities of iPSCs, and their mass differentiation into definitive endoderm and then liver cells,  Because this can potentially provide enough cells to replace a nonfunctional liver, it represents a major step forward in regenerative medicine.