A Link Between Liver Development, Regeneration, and Carcinogensis

The liver has long been recognized as having marked capacity for regeneration. However, it is only lately that we have been able to characterize and define the stem cell populations that contribute to the regeneration of the liver. Several new biomarkers have enabled a new understanding of human hepatic stem cells which has changed the way we think about the relationship between liver development, regeneration, and carcinogenesis. The following is a summary of recent advances in the field of stem cell biology relevant to liver pathology.

Research from the group of Lola M. Reid at the University of North Carolina is bringing the human hepatic stem cells (hHpSC) into clear focus. A study from 2008 published in Hepatology found hHpSC concentrated in the ductal plate during development and restricted to the terminal biliary ducts (canals of hering) in normal adult tissues.  hHpSC are a separate population from the Hepatoblast (HB) which had formerly been regarded as the only stem cell population in the liver. The article defines these two distinct cell populations...

The combination of antigens that uniquely defines hHpSCs (EpCAM, NCAM, CK19, and albumin, but not AFP) is evident in the ductal plates in fetal and neonatal livers and in the canals of Hering in adults. The combination of antigens uniquely defining hHBs (EpCAM, ICAM, CK19, albumin, AFP) is not in cells in the ductal plates but is present in cells throughout the parenchyma of fetal and neonatal livers and in individual cells or small groups of cells connecting to one end of, or adjacent to, a canal of Hering in pediatric and adult livers.

This new perspective allowed the further study of the role these primitive compartments play in the regeneration of human livers. The authors found that the biliary ductular reaction, the putative progenitors that arise from the canals of hering following liver injury, is an expansion of progenitors originating from the hHpSC and HB. Interestingly though, the population that gives rise to the ductular reaction differs depending on the type of injury.

The primary regenerative responses to liver necrosis involve expansion of the hHpSCs (Ep-CAM+, NCAM+, but AFP negative), whereas those in biliary cirrhosis involves presumptive hHBs (EpCAM+at the plasma membrane and ICAM+, AFP+). In hepatic cirrhosis, both populations can be involved.

In a studied carried out by another group, Zhou et al., (Hepatology 2007) showed that a similar set of markers was useful in distinguishing the lineages that the ductular reactions cells contribute to. This study generated beautiful images that depict the ductular reactions becoming bipolarized into hepatocytic and cholangiocytic lineages. An investigation into the transcription factors that are expressed in these ductular reactions confirmed that developmental genes are reactivated and similarly showed differences in expression profiles between injury groups.

I have previously posted on the role of stem cells in tumorigenesis, and hepatic stem cells are a prime suspect for the originating cell of hepatocellular carcinoma (HCC). Last year, Yamashita et al, found an aggressive subset of HCC that contains EpCAM+ cells with the molecular signature of hHpSC. The authors of this study further demonstrated that the EpCAM+ cells are a tumor initiating population and that molecular knockdown of the EpCAM-WNT signalling pathway can attenuate tumor growth. This provides a therapuetics strategy whereby a patient's HCC is assayed for EpCAM expression to determine if anti-EpCAM therapies are indicated. Adecatumumab, an Anti-EpCAM monclonal antibody, has already been used in clinical trials for breast and other cancers. This is an example of a personalized medicine/ targeted molecular therapeutics strategy that is similar to the algorithms now in use for Her2 positive breast cancers and EGFR+/K-Ras wild-type colon cancers.

A pattern is emerging that shows that some tissue stem cells re-express the molecular regulators that govern the embryonic development during regeneration. Further, our new understanding of this link between development and regeneration has opened up new therapeutic areas for cancers that exploit the molecular pathways and mechanisms of the stem cell state for tumor growth, invasions, and metastasis. This is just one area where our pursuit of stem cell research, both embryonic and adult, will lead to new therapeutics for diseases with few, if any, treatments that work.



Zhang L, Theise N, Chua M, & Reid LM (2008). The stem cell niche of human livers: symmetry between development and regeneration. Hepatology (Baltimore, Md.), 48 (5), 1598-607 PMID: 18972441

Zhou H, Rogler LE, Teperman L, Morgan G, & Rogler CE (2007). Identification of hepatocytic and bile ductular cell lineages and candidate stem cells in bipolar ductular reactions in cirrhotic human liver. Hepatology (Baltimore, Md.), 45 (3), 716-24 PMID: 17326146

Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, Reid LM, Minato H, Honda M, Kaneko S, Tang ZY, & Wang XW (2009). EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology, 136 (3), 1012-24 PMID: 19150350

Limaye PB, Alarcón G, Walls AL, Nalesnik MA, Michalopoulos GK, Demetris AJ, & Ochoa ER (2008). Expression of specific hepatocyte and cholangiocyte transcription factors in human liver disease and embryonic development. Laboratory investigation; a journal of technical methods and pathology, 88 (8), 865-72 PMID: 18574450

Obama's Ethics Tough On Approval of New Stem Cell Lines

On March 9, 2009, President Barack Obama issued Executive Order 13505, entitled Removing Barriers to Responsible Scientific Research Involving Human Stem Cells.  This was the order that over turned the much maligned Bush restrictions on federally funding human embryonic stem cells research.  Most researchers in the field thought that better times had come, and in most instances they have.  However, The NIH was mandated with the task of setting forth ethical guidelines that new stem cell lines must meet in order to receive federal funds.  This has much to do with the patient informed consent process, a sometimes tricky ordeal of getting the proper verbiage and language in a signed document, and not with the actual science of stem cells.

The USA Today has run a great article on how tough these ethical guidelines are to meet in some instances and what the cost to science is for those lines that do not meet the standards.

For a decade, Oleg Verlinsky and colleagues at Chicago's Regenerative Genetics Institute created human embryonic stem cells marked with these diseases and others — made from embryos donated by suffering families — hoping to combat these illnesses.

On Thursday, A National Institutes of Health panel ruled one sentence of legal language in the consent form used by RGI meant these hundreds of cell "lines", or colonies, shouldn't receive federal research funding. "They will remain frozen, or discarded, forever," Verlinsky says. "Without federal support, no one will use them for research."

The trouble came from a sentence at the end [of the patient consent form], "We further agree that we, our heirs, successors, relatives, representatives, and/or agents will not bring any action in law or in equity, or in any administrative setting, related to our participation in this study."

That's "exculpatory" language, which waives a patient's rights to sue for negligence or harm, 4 of the 5 ACD panel members agreed, something forbidden under the federal "Common Rule" governing research. One panel member suggested the sentence only applied to lawsuits over profits from any "Patents and Discoveries" made from the cells, but was outvoted.

"There were some enterprising lawyers who probably felt that language needed to be in the consent and didn't appreciate what it might mean," Collins said, at the meeting. "But if we have guidelines, we have to stick to them in order to maintain their credibility." NIH has yet to issue Collins' final decision on the panel's guidance, which recommended approval for the six lines from other institutions.

Many people have asked me lately about the effects of lifting the Bush restrictions and the Obama Administration's support for stem cell research.  While Obama's executive order has been a great thing for the field of stem cell biology and regenerative medicine, It shouldn't be taken to mean that it is now easy to do this research. We can't just go running through the streets doing anything we want with stem cells and human embryos.  It is still a heavily regulated field in which rigorous ethical guidelines must be followed.   Progress is being made, albeit slowly, and we are doing it in an ethical and responsible manner.

California stem cell research: Were voters duped? - latimes.com

Today, the LA Times ran the story California stem cell research: Were voters duped?  It points out the intense criticism that has developed against all the hype surrounding the $3 billion proposition but also acknowledges that science moves slow and the huge challenges that encompass stem cell research. For once the author even calls out the media for hyping the ever caution but optimistic researcher statements.

...the California Institute for Regenerative Medicine, created by that 2004 ballot initiative, has handed out more than $1 billion in research funding. But there have been no "miracles" — no paralyzed people abandoning their wheelchairs or diabetics throwing away their needles. There hasn't even been a human trial of embryonic stem cells, those amazing shape-shifters that can grow into any cell in the body.

So were Californians duped?

Some would say yes. "There have been no cures, no therapies and little progress," Investors Business Daily complained in an editorial earlier this year. Rush Limbaugh went further, declaring embryonic stem cell research "fraudulent, fake."

It's no surprise that the initiative's proponents made big promises: They had something to sell. But instant miracles are uncommon in science, and journalists should do a better job making that clear. We need to highlight the uncertainties in science and, in medical quests such as stem cell therapies, emphasize the baby steps involved that in fact are big leaps: reproducing and growing these flexible cells, understanding how they work, using them to learn about disease, designing treatments and then testing the safety of any resulting therapy.

It will take many more years before we see the fruits of the California endeavor in our clinics, hospital labs, and pharmacies.  There is no question though that this science is some of the most exciting technology that humans have developed.  So, keep in mind, did computers revolutionize our lives overnight?

Breaking down metaplasia

The definition of metaplasia is quite generic - the replacement of one cell type with another.  This implies nothing as to what biological process occurs and our understanding of the mechanisms that convert one cell type to another is immature at best.

There are at least three separate processes that can lead to metaplasia; 

1. Destruction of a cell population which is subsequently replaced by a separate proliferating/migrating cell population, 
2. Respecification of a stem cell or progenitor such that its progeny differentiate into the metaplastic cell types.
3. Direct conversion of a differentiated cell into another without an intermediary, 

The first of these processes, where a proliferating/migrating population fills in for cells that have been destroyed, may be typified by necrotizing sialometaplasia in the minor salivary glands of the oral cavity.  In this pathology the salivary glands undergo necrosis due to injury (i.e. infarction) and the overlying squamous epithelium proliferates down to replace the necrotic glands, filling in where ducts and acini once were. This is shown below.

Diane L. Carlson (2009) Necrotizing Sialometaplasia: A Practical Approach to the Diagnosis. Archives of Pathology & Laboratory Medicine: Vol. 133, No. 5, pp. 692-698

An emerging principle of stem cell biology is that metaplasia may result from the transdetermination of tissue stem cell [1, 2, 3]. Transdetermination is a state transition - the switch from one phenotypic state to another. The state transition concept applies to the processes of differentiation, dedifferentiation, transdifferentiation, and transdetermination as shown below.  Metaplasia may result from various combinations of state transitions.

A stem cell hierarchy is shown in the large box and represents normal differentiation in development and homeostasis. Stem Cells (SC1 and SC2) produce progenitor cells (P 1-3) that then fully differentiate into mature cell types (M1-6). Each step in the normal differentiation pathway is considered a state transition. Transdifferentiation is the transition of one differentiated cell state to another and is proposed to be more likely between closely related cell types (M1 to M2 > M1 to M3). Dedifferentiation is the transition from a more differentiated state to a less differentiated one (M1 to P1). Transdetermination is the transition from one undifferentiated state (SC1 or P2) to a more differentiated state that is not found in the normal developmental or homeostatic differentiation pathway. Transdetermination events are consistent with metaplasia.

Some types of metaplasia may indeed be due to transdetermination. Intestinal metaplasia of the esophagus is thought to arise by this route.  It is believed that a stem cell or progenitor resides in the ducts of the esophageal submucosal glands and facilitates the regeneration of the esophageal epithelium following reflux injury. The contents of the reflux are suspected to cause the transdetermination into the intestinal lineages.

Transdifferentiation, the direct conversion of a differentiated cell type into another, can be achieved experimentally by inducing expression of transcriptional regulators or growth factors.  Reprogramming pancreatic acinar cells by the expression of developmentally regulated genes such as Pdx-1, Ngn3, and Mafa has demonstrated that these mature cell types can be transdifferentiated into beta cells.  This is one of many experimental scenarios where developmentally related cell types can be interconverted. Reprogramming somatic cells is a hot topic in the regenerative medicine field and will be a feature of upcoming posts.

Are there other mechanisms of metaplasia? Post your ideas!

References
1. Eberhard, D. and D. Tosh, Transdifferentiation and metaplasia as a paradigm for understanding development and disease. Cell Mol Life Sci, 2008. 65(1): p. 33-40.
2. Tosh, D. and J.M. Slack, How cells change their phenotype. Nat Rev Mol Cell Biol, 2002. 3(3): p. 187-94.
3. Zhou, Q. and D.A. Melton, Extreme makeover: converting one cell into another. Cell Stem Cell, 2008. 3(4):

Stem cell biology challenges traditional view of tumorigenesis

A very interesting aspect of stem cell biology is the hypothesis that stem cells and progenitors may be the origin of some cancers.  Below is an excerpt from the research group web page of Dr. Eva Hernando.  I believe this sums up the current views on this subject quite nicely.

Traditionally, mature cells in specific tissues and organs have been regarded as the cell-of-origin of the corresponding tumors. However, the observation that tumor cells need to accumulate genetic and phenotypic alterations over extended time periods has turned the view in recent years to stem cells or progenitors with a prolonged lifespan, broadly distributed in local reservoirs [1]. These cells, in charge of maintaining tissue homeostasis, are now considered as the potential target of neoplastic transformation.

Although this notion has been largely accepted for certain leukemias and lymphomas in which the corresponding hematopoietic progenitors are precisely defined, this theory has not yet permeated the field of solid tumors, with few exceptions [2-4]. The identification of maturation stages in non-hematopoietic (i.e., epithelial, mesenchymal) cell types is proving difficult due to the lack of distinctive markers and the low abundance of such intermediates in adult tissues, except after trauma-induced regeneration.

Our laboratory is studying whether certain sarcomas originate from mesenchymal progenitors [5, 6] and whether melanomas result from transformation of melanocytic precursors [7, 8]. Moreover, we hypothesize that alterations in the normal differentiation process of these progenitors act at early stages of tumor initiation, and that the retention or reactivation of stem cell properties may contribute to tumor progression and aggressive behavior (resistance to therapy, metastasis). A limitation for these studies is our partial understanding of the normal differentiation process of these two lineages.

The stem cell state and the process of differentiation are regulated in part by signalling pathways, many of which are a function of kinase activity.  Will we be able to target these stem cell specific pathways with small molecule inhibitors to prevent cancers?

Primer on stem cell biology

Questions such as "So what makes this a stem cell?" and "what's the difference between progenitors and stem cells anyway?" have been asked to me before by both attending physicians and residents or fellows.   Stem cell biology and regenerative medicine is truly a multidisciplinary field and one of the greatest advances in science and technology.  This post is a primer on stem cell biology focuses mostly on adult tissue stem cells and is intended for physicians interested in becoming involved in regenerative medicine.  The following terms are a necessary starting point for discussing the facets that underlie this field and over time I intend to expand upon the concepts presented here.

Stem cells are inherently different from terminally differentiated cells. They posses the capacity for self renewal - the ability maintain an undifferentiated state through cellular divisions.  Many molecular factors have been elucidated that regulate this process such as growth factors (WNT and FGF) and transcription factors (LGR5 and Ascl2 in the intestinal crypt stem cell).  Self renewal is typically proven by animal model lineage studies or through in vitro culture and colony forming assays for isolated human tissues.  Stem cells in adult tissues are mostly quiescent which, experimentally speaking, gives rise to the term label retaining cell.  Controlling the factors that regulate self renewal is currently a major problem slowing the development of regenerative cellular therapeutics and an area of intensive research.

Watch Irv Weissman, director of Stanford's Stem Cell Biology and Regenerative Medicine Institute, speak about the differences between adult and embryonic stem cells.

A progenitor is a cell that is not fully differentiated and thus has an immature phenotype or function. Progenitors may exist in many forms before becoming fully mature and differentiated.  Progenitors are usually the result of an asymmetric stem cell division; hence, they are progeny that are not maintained in an undifferentiated state. This occurs as a coordinated process between extrinsic factors, such as basement membrane components or growth factors and intrinsic regulators such as transcription factors. A population of these progenitors is often referred to as a transit amplifying compartment due to their marked capacity for proliferation.

Take for example the stem cells that reside in the crypts of the intestinal epithelium (shown below). The surface epithelial cells have a finite lifespan and will be sloughed off into the lumen when they are too old and senescent to function. The stem cells in the crypts, however, exist for the lifetime of the organism due to their capacity for self renewal.

from http://www.pnas.org/content/104/39/15418/F5.expansion.html

I have emphasized the word "usually" in the above definition since progenitor populations may not be so well defined, especially during disease processes and tissue regeneration.  The result of one of these processes may be the emergence or induction of a facultative progenitor from cells other than classically defined stem cells or transit amplifying cells.  Furthermore, a population of stem cells may be reserved for mediating the process of regeneration that follows only after severe injury. These reserve stem cells may reside in a completely separate microanatomical compartment which may serve to protect them from injury. When considering this population, we can make a distinction between these reserve stem cells and homeostatic stem cells such as those in the intestinal crypts.  This will be critical when examining the role of progenitors and stem cells in disease processes such as metaplasia and neoplasia.  Future posts will focus on this topic as it relates to both adult and embryonic stem cell studies.

See the Wikipedia article on stem cells that includes many other terms relevant to lineages and potency.