Repopulation of Damaged Livers With Skin-Derived Stem Cells

Patients with severe liver disease must receive a liver transplant. This major procedure requires that the patient survives major surgery and then takes anti-rejection drugs for the rest of their lives. In general, liver transplant patients tend to fair pretty well. The one-year survival rate of liver transplant patients approaches 90% (see O’Mahony and Goss, Texas Heart Institute Journal 2012 39(6): 874-875).

A potentially better way to treat liver failure patients would be to take their own liver cells, convert them into induced pluripotent stem cells (iPSCs), differentiate them into liver cells, and use these liver cells to regenerate the patient’s liver. Such a treatment would contain a patient’s own liver cells and would not require anti-rejection drugs.

Induced pluripotent stem cells or iPSCs are made from genetically-engineered adult cells that have had four specific genes (Oct4, Klf4, Sox2, and c-Myc) introduced into them. As a result of the heightened expression of these genes, some of the adult cells dedifferentiate and are reprogrammed into cells that resemble embryonic stem cells. Normally, this procedure is relatively inefficient, slow, and induces new mutations into the engineered cells. Also, when iPSCs are differentiated into liver cells (hepatocytes), they do not adequately proliferate after differentiation, and they also fail to properly function the way adult hepatocytes do.

New work from laboratories at the University of California, San Francisco (UCSF), has differentiated human hepatocytes by means of a modified technique that bypasses the pluripotency stage. These cells were then used to repopulate mouse livers.

“I really like this paper. It’s a step forward in the field,” said Alejandro Soto-Gutiérrez, assistant professor of pathology at the University of Pittsburgh, who was not involved in the work. “The concept is reprogramming, but with a shortcut, which is really cool.”

Research teams led by Holger Willenbring and Sheng Ding isolated human skin cells called fibroblasts and infected them with engineered viruses that forced the expression of three genes: OCT4, SOX2, and KLF4. These transduced cells were grown in culture in the presence of proteins called growth factors and small molecules in order to induce reprogramming of the cells into the primary embryonic germ layer known as endoderm. In the embryo, the endoderm is the inner-most layer of cells that forms the gastrointestinal tract and its associated structures (liver, pancreas, and so on). Therefore, the differentiation of adult cells into endodermal progenitor cells provides a handy way to form a cell type that readily divides and can differentiate into liver cells.

“We divert the cells on their path to pluripotency,” explained coauthor Holger Willenbring, associate professor of surgery at UCSF. “We still take advantage of what is intrinsic to reprogramming, that the cells are becoming very plastic; they’ve become flexible in what kind of cell type they can be directed towards.”

The authors called these cells induced multipotent progenitor cells (iMPCs). The iMPCs were easily differentiated into endodermal progenitor cells (iMPC-EPCs). These iMPC-EPCs were grown in culture with a cocktail of small molecules and growth factors to increase iMPC-EPC colony size while concomitantly maintain them in an endodermal state. Afterwards, Willenbring and others cultured these cells with factors and small molecules known to promote liver cell differentiation. When these iMPC-Hepatocytes (Heps) were transplanted into mice with damaged livers, the iMPC-Hep cells continued to divide at least nine months after transplantation. Furthermore, the transplanted cells matured and displayed gene expression profiles very similar to that of typical adult hepatocytes. Transplantation of iMPC-Heps also improved the survival of a mouse model of chronic liver failure about as well as did transplantation of adult hepatocytes.

“It is a breakthrough for us because it’s the first time that we’ve seen a cell that can actually repopulate a mouse’s liver,” said Willenbring. Willenbring strongly suspects that iMPCs are better able to repopulate the liver because the derivation of iMPC—rather than an iPSC—eliminates some steps along the path to generating hepatocytes. These iMPCs also possess the ability to proliferate in culture to generate sufficient quantities of cells for therapeutic purposes and, additionally, can functionally mature while retaining that proliferative ability to proliferate. Both of these features are important prerequisites for therapeutic applications, according to Willenbring.

Before this technique can enter clinical trials, more work must be done. For example: “The key to all of this is trying to generate cells that are identical to adult liver cells,” said Stephen Duncan, a professor of cell biology at Medical College of Wisconsin, who was not involved in the study. “You really need these cells to take on all of the functions of a normal liver cell.” Duncan explained that liver cells taken directly from a human adult might be able to repopulate the liver in this same mouse model at levels close to 90 percent.

Willenbring and his colleagues observed repopulation levels of 2 percent by iMPC-Heps, which is substantially better than the 0.05 percent repopulation typically accomplished by hepatocytes derived from iPSCs or embryonic stem cells. However: “As good as this is, the field will need greater levels of expansion,” said Ken Zaret of the Institute for Regenerative Medicine at the University of Pennsylvania, who did not participate in the work. “But the question is: What is limiting the proliferative capacity of the cells?”

Zaret explained that it is not yet clear whether some aspect of how the cells were programmed that differed from how they normally develop could have an impact on how well the population expands after transplantation. “There still is a ways to go [sic],” he said, “but [the authors] were able to show much better long-term repopulation with human cells in the mouse model than other groups have.”

See S. Zhu et al., “Mouse liver repopulation with hepatocytes generated from human fibroblasts,” Nature, doi:10.1038/nature13020, 2014.

Bmi1 Controls Adult Stem Cell “Stemness”

Stem cell scientists from the laboratory of Ophir Klein at UC San Francisco have discovered a new role for a protein called Bmi1 that might give clues as to how to get adult stem cells to regenerate organs.

Ophir Klein, the director of the Craniofacial and Mesenchymal Biology Program and chairman of the Division of Craniofacial Anomalies at UC San Francisco, said “Scientists have known that Bmi1 is a central control switch within the adult stem cells of many tissues, including the brain, blood, lung and mammary gland. Bmi1 also is a cancer-causing gene that becomes reactivated in cancer cells.”

Crystal structure of the BMI1 protein
Crystal structure of the BMI1 protein

All stem cells are somewhat immature in comparison to their adult counterparts. Stem cells also have the capacity to divide almost indefinitely and generate specialized cells. Bmi1 acts as a molecular switch that, if pushed in one direction, drives stem cells to proliferate and grow, but if pushed in the opposite direction, keeps cell proliferation in check. Research from Klein’s lab now suggests that Bmi1 might prevent the progeny of stem cells from differentiating into the wrong cell types in the wrong location.

Downstream targets of Bmi1
Downstream targets of Bmi1

This new discovery suggests that manipulation of Bmi1 and other regulatory molecules might be some of the steps included in laboratory recipes to turn specialized cell development on and off to create new cell-based treatments for tissue lost to injury, disease, or aging.

Also, the dual role of Bmi1 in pathological settings might be intriguing. Cancers are, in many cases, driven by adult stem cells that behave abnormally. If these stem cells could be differentiated, then their growth would slow. Possibly, inactivating Bmi1 in tumor stem cells might be one strategy.

In these experiments, Klein and his colleagues examined those adult stem cells found in the large incisors of mice. Unlike humans, these teeth grow continuously and are, therefore, an attractive model for stem cell research. Klein explained, “There is a large population of stem cells, and the way the daughter cells of the stem cells are produced is easy to track – it’s if they are on a conveyor belt.” Early in life, human beings possess a stem cell population that similarly drive tooth development, but they become inactive after the adult teeth are fully formed during early childhood.

Mouse mandible showing  the large, paired incisors
Mouse mandible showing the large, paired incisors

In the current study, postdoctoral research fellows Brian Biehs and Jimmy Hu showed that at the base of the growing mouse incisor there is a stem cell population that actively expresses Bmi1. In these cells, Bmi1 suppressed a set of genes called Hox genes. When activated, the Hox genes trigger the development of specific cell types and body structures.

In the mouse incisor, Bmi1 keeps these stem cells in their stem cell state and prevent them from differentiating prematurely or inappropriately. “This new knowledge is useful in a fundamental way for understanding how cell differentiating is controlled and may help us manipulate stem cells to get them to do what we want to do,” said Klein.

As they state in the abstract of their paper: “As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.”

Thus this finding from the Klein lab may provide a vital clue for the manipulation of adult stem cells and, perhaps, cancer cells.

Human Brain Cells Made in the Lab that Grow in the Mouse Brain

The laboratory of Arnold Kriegstein, who serves as the director of the Broad Center of Regenerative Medicine and Stem Cell Research at UC San Francisco has made a vitally important type of brain cell from human pluripotent stem cells that smoothly integrates into the brains of laboratory animals. Such a discovery could potentially provide cells that could treat epileptics, Parkinson’s disease or even Alzheimer’s disease.

Medial ganglionic eminence (MGE) cells are a unique type of progenitor cell in the developing brain that guides cell and axon migration. The MGE is located between the thalamus and the caudate nucleus in the developing brain and it facilitates tangential cell migration during embryonic development in the brain.

In the developing brain, cells move radially, along special glial cells that act as tracts for the migrating cells, or tangentially between the radial glial cells. Those cells that move tangentially (perpendicular to the radial glial cells) are specially designated to form GABAminergic neurons; that is neurons that use gamma-amino-butyric acid as their neurotransmitter. However, the MGE also contributes cells to the basal ganglia, which helps control voluntary movement, and guides those axons that grow from the thalamus into the cerebral cortex, or, conversely, those axons that grow from the cerebral cortex to the thalamus. The MGE is a transient structure, and after one year of age, the MGE disappears.

medial ganglionic eminence

Making MGE cells from pluripotent stem cells has been one of the holy grails of developmental neurobiology. Now Kirgstein’s laboratory has succeeded in doing just that.   By subjecting human embryonic stem cells and induced pluripotent stem cells to a complex and extensive differentiation procedure, Kirgstein and his coworkers succeeded in producing large quantities of MGE progenitors that readily matured into forebrain interneurons.  They treated pluripotent stem cells with several growth factors, but more importantly, they timed the delivery of these factors to shape their developmental path.  By conducting neurophysiological experiments on the cells as they differentiated them, Kirgstein and coworkers discovered that they could effectively determine if they had properly derived GABAminergic interneurons.  Jiadong Chen in Kirgsteins’s laboratory showed that the MGE-like progenitors formed proper synapses or connections with other neurons and responded appropriately when stimulated.  Also, as the interneurons matured into more adult-like interneurons, their neurophysiology became more adult-like.  

When grown in the laboratory in culture or when injected into the brains of mice, these MGE-like cells developed into GABAergic interneuron subtypes that displayed the properties of mature GABAminergic neurons.  Also, the cells kept these properties for up to 7 months, and therefore, faithfully mimicked endogenous human neural development.

When injected into mouse brains, the MGE-like progenitors integrated into the brain and formed connections with existing cells.  According to Kirgstein, this property of these cells and their behavior in living tissue makes them prime candidates to test interneuron malfunction that is characteristic of human diseases.  They might also provide material to treat patients who suffer from neurological diseases that affect interneuron function.

According to Kirgstein, “We think that this one type of cell may be useful in treating several types of neurodevelopmental and neurodegenerative disorders in a targeted way.”

In earlier work, Kirgstein implanted mouse MGE cells into the spinal cord of mice that suffered from neuropathic pain.  The implanted cells reduced the pain of those mice, suggesting that they can be used to treat other neurological conditions such a spasticity, Parkinson’s disease and epilepsy.

The first author of this paper, Cory Nicholas said, “The hope is that we can deliver these cells to various places within the nervous system that have been overactive and that they will functionally integrate and provide regulated inhibition.”