Category Archives: Ohio Stem Cells

Doug Lederman is editor and co-founder of Inside Higher Ed. He helps lead the news organization's editorial operations, overseeing news content, opinion pieces, career advice, blogs and other features. Doug speaks widely about higher education, including on C-Span and National Public Radio and at meetings and on campuses around the country, and his work has appeared in The New York Times and USA Today, among other publications. Doug was managing editor of The Chronicle of Higher Education from 1999 to 2003. Before that, Doug had worked at The Chronicle since 1986 in a variety of roles, first as an athletics reporter and editor. He has won three National Awards for Education Reporting from the Education Writers Association, including one in 2009 for a series of Inside Higher Ed articles he co-wrote on college rankings. He began his career as a news clerk at The New York Times. He grew up in Shaker Heights, Ohio, and graduated in 1984 from Princeton University. Doug lives with his wife, Kate Scharff, in Bethesda, Md.

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Making Blood Stem Cells on a Microchip: Academic Minute

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Novel detection of stem cell niche within the stroma of limbus in the rabbit during postnatal development | Scientific Reports - Nature.com

The U.S. Air Defense the Air Force, National Guard and reserves has a 97% COVID-19 vaccination rate.For the remaining 3% without exemptions the time to get the shot is now.Many from the Metro are refusing on religious grounds and risking their careers in the process.In August, the U.S. Military's mandatory vaccination policy will turn one year old. Now it's turned into a fight in the courtroom with dozens saying not only is the mandate unfair it's illegal.For Kent Snider, the hat on his head isn't just a symbol of patriotism."I love the Air Force. I don't want to leave." Snider said.It's a way of life and a way to provide for his family."I do everything for them," Snider said.The technical sergeant is emotional because that way of life could soon end."My next step is separation orders. So it's been a pretty rough couple of months," Snider said.He's one of 36 airmen who've filed a joint lawsuit in the 8th U.S. Circuit Court of Appeals. They've all refused the vaccine. More than half are stationed at Offutt Air Force Base, with a majority of the others at McConnell Air Force Base in Wichita, Kan. The suit says: "The Airmen are now being denied the very liberty they pledged to protect. Each has a sincere religious objection to receiving a COVID-19 vaccination." Of the 36 men in this litigation, 25 have received initial denials, and 22 of the 25 have had their appeals denied. "It has no place in the United States of America where we founded this country on religious freedom," Kris Kobach, the attorney who represents the airmen, said.According to the suit, preliminary injunctive relief is warranted because "the Airmen are likely to prevail on their merits, they are suffering irreparable injury, and the balancing of the equities favors a preliminary injunction." Of the 9,665 processed religious exemption requests in the U.S. Air Force, 118, or about 1.2%, have been approved."They've granted a handful to people, but only ones who're already leaving the Air Force. They haven't granted a single one purely based on the religious exemption request," Kobach said.KETV NewsWatch 7's Bill Schammert asked UNMC Dr. Mark Rupp: "A lot of these military members are citing deeply held religious views going back to the research and development of vaccines that fetal or stem cells were used. Is that true?""The mRNA vaccines in their very early developmental phase did have proof of effectiveness testing with some fetal cells," Rupp said.But Rupp is also quick to point out that leaders in all major religions have backed the COVID-19 vaccine."During a pandemic, I think all of us need to pull together for the common good. The question of whether mandates work? Clearly they work," Rupp said."It's my belief that it changes my body too much and more than God intended," Master Sgt. Josh Welter said.He's ready to sacrifice 15 years of active duty, financial stability for his family and retirement pay to fight for what he believes in.According to the suit, the Airmen have stated they'll accept whatever testing, isolation, remote work, social distancing, masking, or other requirements the Air Force imposes upon them as a condition of them remaining unvaccinated. They're seeking the same scope of treatment as those who've received medical exemptions. As of June 2022 statistics, there have been 676 medical exemptions granted in the Air Force. "The process we're taking is a legal, ethical, and moral approach. How come they're not genuinely hearing us out?" Welter said.They will be heard by a judge, but not until September. Whether they'll still be part of the Air Force then is unclear."If I'm told tomorrow 'hey, sorry about your luck, you're done.' That's a big hit. I'll have to find a job somewhere," Snider saidRight now they have July 29 circled on the calendar. That's when an injunction for a separate but similar case in Ohio runs out. Any day after that, they could get a final notice of forced separation.As for their case, Kobach told KETV NewsWatch 7 that ultimately he believes this will end up at the supreme court.The Department of Defense told KETV NewsWatch 7 that it doesn't respond to ongoing litigation.The Nebraska Air National Guard told KETV NewsWatch 7 that their current vaccination rate is 93%."National Guard members must be ready to serve at any time, in places throughout the world, including where vaccination rates are low and disease transmission is high."Gov. Pete Ricketts also told KETV NewsWatch 7 that he supports the airmen."What we're doing, what I've asked General (Daryl) Bohac to do is to be as accommodating as possible to any of our Guard members who have religious or medical exemption. We're working through that right now; trying to find them jobs that don't require deployment," Ricketts said.

The U.S. Air Defense the Air Force, National Guard and reserves has a 97% COVID-19 vaccination rate.

For the remaining 3% without exemptions the time to get the shot is now.

Many from the Metro are refusing on religious grounds and risking their careers in the process.

In August, the U.S. Military's mandatory vaccination policy will turn one year old. Now it's turned into a fight in the courtroom with dozens saying not only is the mandate unfair it's illegal.

For Kent Snider, the hat on his head isn't just a symbol of patriotism.

"I love the Air Force. I don't want to leave." Snider said.

It's a way of life and a way to provide for his family.

"I do everything for them," Snider said.

The technical sergeant is emotional because that way of life could soon end.

"My next step is separation orders. So it's been a pretty rough couple of months," Snider said.

He's one of 36 airmen who've filed a joint lawsuit in the 8th U.S. Circuit Court of Appeals. They've all refused the vaccine. More than half are stationed at Offutt Air Force Base, with a majority of the others at McConnell Air Force Base in Wichita, Kan.

The suit says: "The Airmen are now being denied the very liberty they pledged to protect. Each has a sincere religious objection to receiving a COVID-19 vaccination."

Of the 36 men in this litigation, 25 have received initial denials, and 22 of the 25 have had their appeals denied.

"It has no place in the United States of America where we founded this country on religious freedom," Kris Kobach, the attorney who represents the airmen, said.

According to the suit, preliminary injunctive relief is warranted because "the Airmen are likely to prevail on their merits, they are suffering irreparable injury, and the balancing of the equities favors a preliminary injunction."

Of the 9,665 processed religious exemption requests in the U.S. Air Force, 118, or about 1.2%, have been approved.

"They've granted a handful to people, but only ones who're already leaving the Air Force. They haven't granted a single one purely based on the religious exemption request," Kobach said.

KETV NewsWatch 7's Bill Schammert asked UNMC Dr. Mark Rupp: "A lot of these military members are citing deeply held religious views going back to the research and development of vaccines that fetal or stem cells were used. Is that true?"

"The mRNA vaccines in their very early developmental phase did have proof of effectiveness testing with some fetal cells," Rupp said.

But Rupp is also quick to point out that leaders in all major religions have backed the COVID-19 vaccine.

"During a pandemic, I think all of us need to pull together for the common good. The question of whether mandates work? Clearly they work," Rupp said.

"It's my belief that it changes my body too much and more than God intended," Master Sgt. Josh Welter said.

He's ready to sacrifice 15 years of active duty, financial stability for his family and retirement pay to fight for what he believes in.

According to the suit, the Airmen have stated they'll accept whatever testing, isolation, remote work, social distancing, masking, or other requirements the Air Force imposes upon them as a condition of them remaining unvaccinated.

They're seeking the same scope of treatment as those who've received medical exemptions. As of June 2022 statistics, there have been 676 medical exemptions granted in the Air Force.

"The process we're taking is a legal, ethical, and moral approach. How come they're not genuinely hearing us out?" Welter said.

They will be heard by a judge, but not until September. Whether they'll still be part of the Air Force then is unclear.

"If I'm told tomorrow 'hey, sorry about your luck, you're done.' That's a big hit. I'll have to find a job somewhere," Snider said

Right now they have July 29 circled on the calendar. That's when an injunction for a separate but similar case in Ohio runs out. Any day after that, they could get a final notice of forced separation.

As for their case, Kobach told KETV NewsWatch 7 that ultimately he believes this will end up at the supreme court.

The Department of Defense told KETV NewsWatch 7 that it doesn't respond to ongoing litigation.

The Nebraska Air National Guard told KETV NewsWatch 7 that their current vaccination rate is 93%.

"National Guard members must be ready to serve at any time, in places throughout the world, including where vaccination rates are low and disease transmission is high."

Gov. Pete Ricketts also told KETV NewsWatch 7 that he supports the airmen.

"What we're doing, what I've asked General (Daryl) Bohac to do is to be as accommodating as possible to any of our Guard members who have religious or medical exemption. We're working through that right now; trying to find them jobs that don't require deployment," Ricketts said.

See the original post here:
Why Nebraska's U.S. Airmen have filed a lawsuit refusing the COVID-19 vaccine - KETV Omaha

J Clin Invest. 2013 May 1; 123(5): 19021910.

1Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Liver Center and Diabetes Center, Institute for Regeneration Medicine, and 2Department of Pathology, UCSF, San Francisco, California, USA. 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

1Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Liver Center and Diabetes Center, Institute for Regeneration Medicine, and 2Department of Pathology, UCSF, San Francisco, California, USA. 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

1Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Liver Center and Diabetes Center, Institute for Regeneration Medicine, and 2Department of Pathology, UCSF, San Francisco, California, USA. 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

1Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Liver Center and Diabetes Center, Institute for Regeneration Medicine, and 2Department of Pathology, UCSF, San Francisco, California, USA. 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

1Department of Biochemistry and Biophysics, Programs in Developmental and Stem Cell Biology, Genetics and Human Genetics, Liver Center and Diabetes Center, Institute for Regeneration Medicine, and 2Department of Pathology, UCSF, San Francisco, California, USA. 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.

Authorship note: Chunyue Yin and Kimberley J. Evason contributed equally to this work.

Hepatic stellate cells are liver-specific mesenchymal cells that play vital roles in liver physiology and fibrogenesis. They are located in the space of Disse and maintain close interactions with sinusoidal endothelial cells and hepatic epithelial cells. It is becoming increasingly clear that hepatic stellate cells have a profound impact on the differentiation, proliferation, and morphogenesis of other hepatic cell types during liver development and regeneration. In this Review, we summarize and evaluate the recent advances in our understanding of the formation and characteristics of hepatic stellate cells, as well as their function in liver development, regeneration, and cancer. We also discuss how improved knowledge of these processes offers new perspectives for the treatment of patients with liver diseases.

Hepatic stellate cells are located in the space of Disse between the sinusoidal endothelial cells and hepatic epithelial cells, and account for 5%8% of the cells in the liver. In a healthy liver, stellate cells are quiescent and contain numerous vitamin A lipid droplets, constituting the largest reservoir of vitamin A in the body (reviewed in ref. 1). When the liver is injured due to viral infection or hepatic toxins, hepatic stellate cells receive signals secreted by damaged hepatocytes and immune cells, causing them to transdifferentiate into activated myofibroblast-like cells (reviewed in ref. 2). As the primary extracellular matrixproducing (ECM-producing) cells in liver, activated stellate cells generate a temporary scar at the site of injury to protect the liver from further damage. In addition, hepatic stellate cells secrete cytokines and growth factors that promote the regeneration of hepatic epithelial cells. In chronic liver disease, prolonged and repeated activation of stellate cells causes liver fibrosis, as characterized by widespread scar formation and perturbation of liver architecture and function (reviewed in ref. 3). Recent clinical and experimental evidence indicates that hepatic fibrosis is reversible upon removal of the underlying etiological agent (46). During the regression of liver fibrosis, the number of activated hepatic stellate cells is greatly reduced by the induction of cellular senescence and apoptosis, or by the return to the quiescent state (2, 57). Because of their pivotal roles in liver repair and disease pathogenesis, hepatic stellate cells have been a major focus of liver research. However, our knowledge of their cell biology is far from complete, mainly due to the challenges of studying these cells in vivo.

This Review focuses on the recent insights and emerging investigations into the formation of hepatic stellate cells and their function in liver development, regeneration, and hepatocellular carcinoma (HCC). The regulation of stellate cells in liver fibrosis as well as the design of antifibrotic therapies is reviewed separately in this issue (8).

Over the past two decades, the development of cell culture system and genetic animal models (summarized in Figure ) has greatly advanced our understanding of the cellular properties of hepatic stellate cells and their function in healthy as well as injured livers. When cultured on plastic, freshly isolated hepatic stellate cells undergo spontaneous activation (911). This cell culture system, along with other hepatic stellate cell lines (1214), recapitulates many aspects of hepatic stellate cell activation in vivo. But hepatic stellate cells activated in culture do not fully reproduce the changes in gene expression observed in vivo, making it difficult in some cases to correlate in vitro results with hepatic stellate cell behaviors in vivo (15).

(A) Phase contrast image of mouse hepatic stellate cells cultured for 2 days. These hepatic stellate cells are still quiescent, as evidenced by their vitamin A lipid deposition, a stellate morphology, and presence of dendritic processes. (B) Phase contrast image of mouse hepatic stellate cells cultured for 14 days. By this time, hepatic stellate cells are fully activated and exhibit dramatic changes in their morphology and reduction in lipid deposition. (C) Fluorescence image of hepatic stellate cells in healthy adult mouse liver stained for desmin. (D) Fluorescence image shows -SMA immunostaining in CCl4-induced fibrosis in the adult mouse liver. (E) Confocal single-plane image of Tg(hand2:EGFP) expression in zebrafish hepatic stellate cells at 5 days after fertilization. The hepatic stellate cells exhibit a stellate morphology and send out complex protrusions (23). (F) Confocal single-plane image of hepatic stellate cells labeled by Tg(hand2:EGFP) expression in zebrafish larvae treated with 2% ethanol from 4 to 5 days after fertilization. Hepatic stellate cells become activated upon the acute ethanol assault, as evidenced by the loss of complex cellular processes and elongated cell body, suggestive of changes in contractility (24).

In the animal, hepatic stellate cells can be identified based on expression of desmin (16) and glial fibrillary acidic protein (GFAP) (17) in the quiescent state and -SMA in the activated state (18). The identification of promoters that selectively drive transgene expression in hepatic stellate cells might facilitate both in vivo observations and genetic manipulation of these cells. Components of collagen 1(I), collagen 2(I), and SMA promoters were used to direct reporter gene expression in activated hepatic stellate cells in transgenic mice (19). Promoter elements of the Gfap (20, 21) and vimentin (6) genes drive gene expression in quiescent hepatic stellate cells. However, neither promoter is specific for hepatic stellate cells: Gfap promoter activity is detected in neuronal tissues and cholangiocytes (21), whereas the vimentin gene is also expressed in vascular smooth muscle cells and portal fibroblasts (6).

The zebrafish has emerged as a valuable vertebrate model system to study liver development and disease. The rapid external development and translucence of zebrafish embryos and larvae make them well suited for in vivo imaging (22, 23). The availability of transgenic lines that express fluorescent proteins in different hepatic cell types allows easy visualization of cell behaviors in the animal and greatly facilitates genetic and chemical screens to identify regulators of liver development and disease pathogenesis. Our group recently reported a transgenic zebrafish line, Tg(hand2:EGFP), that expresses EGFP under the promoter of the hand2 gene (24). The transgene expression marks both quiescent and activated hepatic stellate cells. Zebrafish hepatic stellate cells exhibit all the hallmarks of mammalian hepatic stellate cells, including morphology, localization, vitamin A storage, and gene expression profile. Significantly, zebrafish hepatic stellate cells become activated in response to an acute alcohol insult, as evidenced by increased proliferation and ECM production (Figure and ref. 24). This zebrafish hepatic stellate cell reporter line thus represents a novel animal model that complements the cell culture and mammalian model systems.

Knowledge about the characteristics, lineage, and function of stellate cells during liver development is critical to obtaining a fundamental understanding of hepatic stellate cell activation and their role in liver diseases. Recent studies in animal models and cell culture systems have provided key insights regarding hepatic stellate cells during development, but important gaps remain in our knowledge of this process.

The embryonic origin of hepatic stellate cells is unresolved because they express marker genes of all three germ layers (reviewed in ref. 2). Lineage tracing of the Wilms tumor suppressor geneexpressing (Wt1-expressing) cells and mesoderm posterior 1expressing cells in mice showed that hepatic stellate cells develop from the septum transversumderived mesothelium lining the liver (25, 26), suggestive of a mesodermal origin. On the other hand, stellate cells in the human fetal liver express CD34 and cytokeratin-7/8, connecting them to an endodermal origin (27, 28). Along this theme, hepatic epithelial cells are thought to transdifferentiate into hepatic stellate cells in the injured liver through epithelial-mesenchymal transition (EMT) (29). However, the contribution of EMT to the hepatic stellate cell lineage is highly controversial (30). Lastly, bone marrowderived mesenchymal cells are also thought to contribute to both quiescent and activated hepatic stellate cells (31, 32), although several reports indicate that this contribution is negligible (33, 34).

It is noteworthy that in mice, the septum transversum-derived mesothelial cells give rise not only to hepatic stellate cells, but also to perivascular mesenchymal cells, including portal fibroblasts, smooth muscle cells around the portal vein, and fibroblasts around the central vein (26). Following liver injury, activated stellate cells are the major source of myofibroblasts. However, portal fibroblasts and vascular myofibroblasts can also become myofibroblasts, but their contribution to fibrogenesis might be different from the hepatic stellate cellderived myofibroblasts (35, 36). Therefore, an understanding of how the cell fate decision is made between hepatic stellate cells and perivascular mesenchymal cells might aid in the design of therapies to specifically target hepatic stellate cells.

In both fetal and adult livers, stellate cells are closely associated with sinusoidal endothelial cells, which also derive from mesoderm. Because of their physical proximity and shared expression of angiogenic factors (37), hepatic stellate cells and sinusoidal endothelial cells have been proposed to share a common precursor. This hypothesis is supported by observations in chick embryos that the mesothelium contributes to both cell populations (38). In zebrafish, however, stellate cells are still present in the liver of cloche mutants that lack sinusoidal endothelial cells and their precursors (24). This result indicates that neither endothelial cells nor their precursors are required for hepatic stellate cell differentiation or their entry into the liver.

To date, only a few studies have addressed early hepatic stellate cell behaviors in vivo. Tracking of the Wt1-expressing septum transversum cells in mice showed that these cells migrate inward from the liver surface while differentiating into hepatic stellate cells (ref. 25 and see Figure A). A similar migration behavior of hepatic stellate cells was observed in zebrafish (24). Furthermore, the migration of septum transversum cells from the liver surface likely constitutes the main source of new stellate cells during zebrafish development, as they rarely proliferate after entering the liver.

(A) Hepatic stellate cell development. Lineage-tracing analyses in mice indicate that during development, the mesodermal cells within the septum transversum invade the liver while differentiating into hepatic stellate cells and perivascular mesenchymal cells. VEGF and retinoic acid signaling are both required for hepatic stellate cell formation, potentially affecting the migration of septum transversum cells, the differentiation of hepatic stellate cells, or both. Wt1, Wnt/-catenin signaling, and Lhx2 inhibit aberrant activation of hepatic stellate cells in the developing liver. (B) Contribution of hepatic stellate cells to hepatic organogenesis. The biological processes influenced by hepatic stellate cells are indicated in blue. For endothelial cells, hepatic stellate cells secrete the chemokine SDF1, whereas endothelial cells express its receptor CXCR4. Concurrently, endothelial cells produce PDGF, whereas hepatic stellate cells express its receptor. SDF1 and PDGF signaling maintain the close association between hepatic stellate cells and endothelial cells, which is critical for vascular tube formation and integrity. For hematopoietic stem cells (HSCs), hepatic stellate cells mediate their recruitment to the liver via SDF1/CXCR4 signaling. For hepatic epithelial cells, hepatic stellate cells regulate the proliferation of hepatoblast progenitor cells and hepatocytes by producing growth factors such as Wnt, FGF, HGF, and retinoic acid. They may also modulate the differentiation of hepatocytes and biliary cells from hepatoblasts by controlling the ECM composition within the liver. Lastly, hepatic stellate cells may contribute to the development of biliary cells by expressing the Notch ligand jagged 1 (Jag1).

Studies in mutant mice have revealed the roles of several mesenchymal-specific genes in hepatic stellate cell development (summarized in Figure A). Wt1 and the LIM homeobox gene Lhx2 are both expressed in the septum transversum and hepatic stellate cells during development (26, 39). Wt1-null fetal livers show an abnormal increase of -SMA expression (40), suggestive of ectopic stellate cell activation. Similarly, Lhx2 knockout embryos contain numerous activated hepatic stellate cells and display a progressively increased deposition of ECM proteins associated with fibrosis (41). Therefore, despite being dispensable for hepatic stellate cell formation, both Wt1 and Lhx2 appear to keep these cells quiescent during development. The signal downstream of Wt1 and Lhx2 that prevents hepatic stellate cell activation is unclear. One candidate is the Wnt/-catenin pathway, as conditional deletion of -catenin in the mesenchyme results in increased -SMA expression and ECM deposition in the liver (42, 43). On the other hand, freshly isolated hepatic stellate cells from adult mice exhibit hedgehog (Hh) pathway activity, and inhibition of Hh signaling via pharmacologic inhibitor or neutralizing antibodies to Hh impairs hepatic stellate cell activation and decreases their survival (44). It will be interesting to investigate the role of the Hh pathway during the development of hepatic stellate cells.

Studies of the zebrafish hepatic stellate cell reporter line have shed light on the regulation of their differentiation and migration into the liver. Inhibition of VEGF signaling by global knockdown of VEGFR2 or by treatment with a VEGFR2 pharmacologic inhibitor during the course of hepatic stellate cell differentiation and migration drastically reduces their numbers (24). VEGF signaling does not appear to be essential for hepatic stellate cell survival, as blocking VEGFR2 during later stages only caused a moderate decrease in hepatic stellate cell numbers. Rather, VEGF may be required for hepatic stellate cell differentiation and/or their entry into the liver. Studies of liver injury and cancer have documented VEGF ligand expression by hepatocytes and biliary cells (4547). Likewise, hepatic epithelial cells could be the source of VEGF for hepatic stellate cell development. Using an unbiased chemical screen approach, our group discovered two retinoid receptor agonists that have an opposing effect on hepatic stellate cell development (24). Compounds that modulate stellate cell differentiation, proliferation, or the switch between their quiescent and activated states during development could potentially affect hepatic stellate cell behavior during injury, and thus have direct clinical implications.

Throughout development, hepatic stellate cells are in close proximity to endothelial, hematopoietic, and hepatic epithelial cells, which suggests that hepatic stellate cells may modulate the growth, differentiation, or morphogenesis of these cells (summarized in Figure B). The interactions between stellate cells and other hepatic cells during development could be reactivated when the liver responds to injury.

Hepatic stellate cells contact sinusoidal endothelial cells by means of complex cytoplasmic processes, which ideally positions them for paracrine signaling with endothelial cells (48). During angiogenesis, interactions between pericytes and endothelial cells are essential for vascular tube maturation and integrity (49). Hepatic stellate cells are thought to be the pericyte equivalent in the liver and therefore may have the same impact on the development of the hepatic vasculature (50). In support of this notion, in mice that lack -catenin in the liver mesenchyme, hepatic stellate cells become aberrantly activated and the liver is filled with dilated sinusoids (42).

During mammalian embryogenesis, the liver is the main site of hematopoiesis (51). In mice lacking the hepatic stellate cellexpressing homeobox gene Hlx, fetal liver hematopoiesis is severely impaired (52), implicating hepatic stellate cells in this process. Fetal hepatic stellate cells express stromal cellderived factor 1 (SDF1; also known as CXCL12) (51), a potent chemoattractant for hematopoietic stem cells, which themselves express the SDF1 receptor CXCR4 (53). Therefore, it is plausible that hepatic stellate cells are involved in recruiting hematopoietic stem/progenitor cells into the fetal liver.

Stellate cells first appear in mouse livers at around E10E11, when differentiation of hepatocytes and biliary cells from hepatoblasts is still underway (54). Mouse fetal liver mesenchymal cells promote the maturation of hepatoblasts through cell-cell contact in cell culture (55). In Wt1 and Hlx mutant mice, the hepatoblast population fails to proliferate, resulting in smaller livers (40, 52). Fetal hepatic stellate cells express growth factors and mitogens such as Wnt9a (56), HGF (57), pleiotrophin (58), and FGF10 (59, 60), all of which have profound effects on the proliferation of hepatic epithelial cells during organ development and regeneration. In addition, hepatic stellate cells in the Wt1-null fetal livers show decreased expression of retinaldehyde dehydrogenase 2, an enzyme that catalyzes retinoic acid synthesis (40). The impairment of retinoic acid production could in turn affect hepatoblast proliferation. The role of hepatic stellate cells in hepatoblast differentiation is less clear. Nagai et al. reported that cell-cell contacts between hepatic stellate cells and hepatic epithelial cells induce the differentiation of the hepatocyte fate (61). On the other hand, the emergence and distribution of hepatic stellate cells also seem to correlate with the development of intrahepatic biliary cells (62). Hepatic stellate cells in rats express Notch receptors and target genes of Notch signaling (63), and Notch signaling plays key roles in the differentiation and morphogenesis of intrahepatic biliary cells (64). A recent study showed that inactivation of the Notch ligand jagged 1, which is expressed in the portal vein mesenchyme, leads to a paucity of intrahepatic bile ducts (65). Given that hepatic stellate cells also express jagged 1 (66), it will be interesting to investigate whether they modulate biliary cell development via Notch signaling. Alternatively, hepatic stellate cells could influence hepatoblast differentiation through production of ECM proteins, as different ECM components have different effects on the determination of the hepatocyte and biliary cell fate (67, 68).

The directed differentiation of human pluripotent stem cells into hepatocytes in culture could lead to new cell transplantation therapies for a wide range of acute and chronic liver diseases. Although important progress toward this goal has been made in recent years, liver cells differentiated in vitro do not share all the key characteristics of mature hepatocytes (reviewed in refs. 69, 70). Co-culturing primary human liver progenitor cells or hepatocytes with mesenchymal cells promotes or stabilizes hepatocyte differentiation (7173). Therefore, understanding the interactions between hepatic stellate cells and hepatic epithelial cells during development is essential to create more efficient cell culture protocols for programmed differentiation of stem cells into hepatocytes.

Much as studies of liver development are highly relevant to creating new stem cell therapies, an understanding of liver regeneration has important implications for improving current methods of differentiating and propagating hepatocytes in vitro, as well as for stimulating hepatic recovery and improving survival after acute liver failure, liver transplantation, or resection. One of the oldest and most commonly used rodent models of liver regeneration is partial hepatectomy (PH), in which two-thirds of the animals liver is surgically removed (74, 75). Liver regeneration following PH is mainly driven by replication of existing hepatocytes and occurs in the absence of substantial necrosis and inflammation (74). To model how the liver regenerates when the ability of hepatocytes to divide is compromised, hepatocyte proliferation inhibitors such as 2-acetylaminofluorene can be administered before PH (2AAF/PH), which results in liver repopulation mediated by activation of liver progenitor cells or oval cells rather than proliferation of hepatocytes (74). Other rodent models of liver injury and regeneration involve chemical treatments with carbon tetrachloride (CCl4) or acetaminophen (reviewed in ref. 76) or bile duct ligation (BDL) (77). While the PH model of liver regeneration may be particularly relevant to clinical scenarios in which the quantity of liver tissue is a limiting factor, such as small-for-size syndrome following liver transplantation, chemical injury and BDL models may more faithfully recapitulate the necrosis, inflammation, and/or fibrosis that accompany regeneration in chronic viral hepatitis, biliary tract disease, and/or drug-induced liver injury.

Activated hepatic stellate cells have been implicated in assisting liver regeneration by producing angiogenic factors as well as factors that modulate endothelial cell and hepatocyte proliferation and by remodeling the ECM (78). Recent evidence also suggests that in progenitor cell-mediated liver regeneration, hepatic stellate cells may, through a process of mesenchymal to epithelial transition, give rise to hepatocytes (21). Supporting the involvement of stellate cells in liver regeneration, inhibiting activated hepatic stellate cells using gliotoxin (79) and l-cysteine (80) prevents normal regenerative responses of both hepatocytes and oval cells in acetaminophen and 2AAF/PH-induced liver injuries, respectively. In addition, Foxf1+/ mice subjected to CCl4 injury show decreased hepatic stellate cell activation and more severe hepatocyte necrosis during the regenerative period (81). Notably, the mechanisms by which activated hepatic stellate cells help mediate liver regeneration in human patients and experimental animals remain to be determined and the relative importance of different subtypes of hepatic stellate cells/myofibroblasts is likely to depend on the nature of the initial insult.

Activated hepatic stellate cells produce a wide array of cytokines and chemokines (2). These factors may directly enhance the proliferation of liver progenitor cells and hepatocytes, or they may act indirectly through sinusoidal endothelial cells and immune cells to promote regeneration (ref. 2 and summarized in Figure ). Conditioned media collected from hepatic stellate cells harvested from rats during early liver regeneration following 2AAF/PH injury contain high levels of HGF and promote oval cell proliferation (82). One potential mediator of HGF production by hepatic stellate cells is the neurotrophin receptor P75NTR, which is expressed in human hepatic stellate cells following fibrotic liver injury. Murine hepatic stellate cells deficient for P75NTR do not differentiate properly into myofibroblasts in vitro or following liver injury induced by fibrin deposition in plasminogen-deficient (Plg/) mice (83). Consequently, HGF production and hepatocyte proliferation are impaired in P75NTR;Plg double-mutant mice (83). Hepatic stellate cell differentiation can be restored by constitutively active Rho in P75NTR-deficient hepatic stellate cells in vitro (83). These findings support a model in which P75NTR promotes hepatic stellate cell activation via Rho, and activated stellate cells secrete HGF to stimulate hepatocyte proliferation during regeneration (83). Hh signaling is another important mediator of hepatic stellate cellhepatocyte interactions during regeneration. Culture-activated hepatic stellate cells synthesize sonic hedgehog (Shh), which serves as an autocrine growth factor for these cells (84). In vivo, Hh ligands induce hepatocyte proliferation after PH (85).

The biological processes that are influenced by hepatic stellate cells are indicated in blue. At early phases of liver regeneration, hepatic stellate cells promote the proliferation of liver progenitor cells and hepatocytes. They also stimulate angiogenesis in the wounded area and assist in the recruitment of hematopoietic stem cells and immune cells to the liver (reviewed in ref. 48). Recent studies suggest that activated hepatic stellate cells may undergo a mesenchymal-to-epithelial transition to transdifferentiate into liver progenitor cells. At late phases, hepatic stellate cells participate in the termination of regeneration, likely via high expression of TGF-. Hepatic stellate cells have also been proposed to contribute to HCC development, potentially through dysregulation of some aspects of liver regeneration described above. On the other hand, liver fibrosis, which results from ectopic hepatic stellate cell activation, has controversial roles in HCC. Most evidence suggests that fibrosis promotes HCC, but it is possible that in some clinical settings fibrosis and HCC might occur due to the same underlying factor(s) rather than one promoting the other.

Notably, activated hepatic stellate cells are the main source of matrix metalloproteinases and their inhibitors that participate in ECM remodeling. The production of cytokines and remodeling of the ECM are likely to be coupled, as the ECM is capable of sequestering biologically active molecules (86, 87). Thus in addition to directly secreting cytokines, activated hepatic stellate cells may modulate their function by cleaving or releasing cytokines from the ECM.

Liver regeneration is a multistep process involving both initiation and termination of liver growth. The liver stops regenerating when it attains the mass required for the needs of the organism (88). The most well-known hepatocyte antiproliferative factor is TGF-, and one of the primary TGF-producing cell types in the liver are hepatic stellate cells (89). How do hepatic stellate cells mediate both the initiation and cessation of liver regeneration? As mentioned earlier, conditioned medium collected from hepatic stellate cells at early phases of liver regeneration in a 2AAF/PH injury model contains high levels of HGF. This strong mitogen may override the antiproliferative effect of TGF-1 (82). In contrast, at terminal phases of liver regeneration, hepatic stellate cells produce high levels of TGF-1, which inhibits hepatocyte proliferation and even induces apoptosis. Serotonin has been shown to increase expression of TGF-1 in cultured primary mouse hepatic stellate cells via the 5-hydroxytryptamine 2B (5-HT2B) receptor, and 5-HT2B inhibition promotes hepatocyte proliferation following PH, BDL, and CCl4-induced liver injury (90). Thus, hepatic stellate cells may change their cytokine expression profile during the process of liver regeneration, regulating both its initiation and termination.

To fully characterize the role of hepatic stellate cells in liver regeneration, their specific ablation would be highly useful, ideally at different time points in the regenerative process. While some chemical tools, including gliotoxin (79) or l-cysteine (80), exist for selective inhibition of hepatic stellate cells in rodent models, the possibility that these drugs also affect other hepatic cell types is difficult to exclude. A recent study indicates that hepatic stellate cells can be depleted in mice by using the GFAP promoter to drive the herpes simplex virusthymidine kinase gene expression, rendering proliferating hepatic stellate cells susceptible to gancyclovir-induced death (20). An advantage of this new model is the ability to target proliferating hepatic stellate cells in vivo without affecting quiescent hepatic stellate cells or other myofibroblasts. However, hepatic stellate cells cannot be completely ablated using this model, as GFAP is not universally expressed in these cells.

Any single animal model is unlikely to completely mimic all relevant aspects of human liver regeneration, particularly given that the cellular and molecular pathways mediating regeneration are likely to vary somewhat depending on the nature of the initial injury. Therefore, future studies of hepatic stellate cells in liver regeneration will be facilitated by the availability of multiple animal models, which are likely to yield complementary insights. Advantages of rodent models include the ability to isolate, culture, and activate hepatic stellate cells in vitro, facilitating follow-up cell culture studies focused on molecular mechanisms involved in regeneration. On the other hand, the excellent live-imaging technologies available in zebrafish are well suited for studying the cellular interactions at play during the regenerative process. As with rodents, PH or toxic chemicals can be used to induce liver regeneration in zebrafish (reviewed in ref. 74). Genetic tools have enabled the development of additional regeneration models including the nitroreductase/metronidazole cell ablation system (91) and morpholino-based knockdown of a mitochondrial import gene to induce hepatocyte death (92). One promising approach is to perform high-throughput chemical screens in various zebrafish models of liver injury, seeking drugs that affect stellate cells during liver regeneration (24).

While promotion of hepatocyte proliferation and liver regeneration may be desirable in some clinical settings, aberrant activation of such processes can also be associated with human diseases, most notably HCC (summarized in Figure ). The majority of human HCCs occur in the setting of clinically significant fibrosis or cirrhosis (93), implicating hepatic stellate cells in their pathogenesis as the major ECM-producing cell type of the liver. The associations between HCC and fibrosis are incompletely understood, but likely involve inflammatory cells, integrin signaling, growth factor interactions with the ECM, and communication between activated hepatic stellate cells and tumor cells (reviewed in ref. 94). Activated hepatic stellate cells are present between endothelial cells and cancer cell trabeculae in patients with HCC (95), and conditioned media from activated hepatic stellate cells increases proliferation and migration of human HCC cells (96). Thus, most evidence suggests that fibrosis promotes HCC, but it is possible that in some clinical settings fibrosis and HCC might occur due to the same underlying factor(s) rather than one promoting the other.

Chemical compounds such as N-nitrosodiethylamine, CCl4, and aflatoxin B1 cause HCC in rodents that is preceded by chronic liver injury, mimicking the injury-fibrosis-malignancy sequence that characterizes most human HCCs (97). However, tumor phenotypes in these models are dependent on animal age, strain, and the route of drug administration, and tumor latency can be quite long (97). On the other hand, liver tumors induced genetically in mice via expression of growth factors such as TGF-, oncogenes such as Myc, and viral proteins such as HBX are more tractable but are not usually preceded by substantial fibrosis (98, 99). Thus, the opportunity for studying hepatic stellate cellHCC interactions in transgenic mouse models of HCC has been somewhat limited, with the notable exception of the PDGF-C transgenic mouse (100). These mice, whose hepatocytes express human PDGF-C, show hepatic stellate cell activation and collagen deposition followed by hepatomegaly and HCC. These in vivo findings correlate with in vitro studies demonstrating that PDGF-C promotes the proliferation, survival, and migration of fibroblasts and pericytes (101).

Interactions between hepatic stellate cells and HCC cells in vivo have also been studied by co-transplanting hepatic stellate cells and malignant hepatocytes into immunocompromised mice. These studies have implicated TGF- signaling (102, 103) and regulatory T cells (104) as mechanisms by which hepatic stellate cells may promote HCC growth. On the other hand, experiments performed in lecithin retinol acyltransferasedeficient mice have revealed ways by which HCC growth might be inhibited via targeting of hepatic stellate cells (105, 106). These mice lack retinoid-containing lipid droplets in hepatic stellate cells, exhibit increased retinoic acid signaling, and show decreased tumor formation in response to diethylnitrosamine, suggesting that altering retinoic acid signaling in stellate cells may inhibit HCC growth.

Zebrafish develop liver tumors that are morphologically and genetically similar to human HCC (107110). Similar to many rodent models, zebrafish HCC models are not typically preceded by cirrhosis, although co-expression of hepatitis B virus X and hepatitis C virus core proteins in zebrafish liver leads to fibrosis and cholangiocarcinoma (111). This model may thus be useful to study hepatic stellate cell interactions with primary liver tumor cells in vivo.

While many pathways that mediate hepatic stellate cellHCC interactions have been implicated (reviewed in ref. 94), the effects of specifically inhibiting or activating these pathways in vivo have not been fully explored. Driving expression of candidate positive or negative regulators specifically in hepatic stellate cells or creating stellate cellspecific gene knockouts could be useful in this regard. A major challenge for these experiments, as in studies of hepatic stellate cell development, is the identification of promoters with improved specificity. Similarly, improved techniques for ablating or inhibiting hepatic stellate cells could help tease out the role of these cells at different time points in HCC formation. Such studies could help define when and how hepatic stellate cells could be targeted to prevent or treat HCC.

A more efficient way to detect HCC could profoundly improve prognosis by enabling earlier diagnosis and more effective treatments. New HCC biomarkers that have been proposed include molecules produced by hepatic stellate cells, such as HGF and IGF (112). Patients with HCC also show elevated plasma levels of TGF-1 (113) and osteopontin (114), compared with patients with chronic hepatitis and/or cirrhosis. As many of the same factors are produced by hepatic stellate cells during cirrhosis and during carcinogenesis, it is likely that a combination of biomarkers will be required to optimize early HCC detection.

Studies of hepatic stellate cell behavior during development, regeneration, and tumor formation using cell culture and animal models have provided substantial insights regarding the cellular and molecular mechanisms involved in these processes. It will be crucial to identify promoters with improved cell type specificity, as they will facilitate hepatic stellate cellspecific manipulations, including gene knockouts and cell ablation. Given the critical roles that hepatic stellate cells play in diverse aspects of liver pathophysiology, this intriguing cell type represents a major, and mostly untapped, potential reservoir for the development of therapies targeting a wide variety of human liver diseases, ranging from acute liver failure to drug-induced liver injury to HCC.

The authors thank Jacquelyn Maher for her critical comments and support. C. Yin is supported by grant K99AA020514 from the NIH and the University of California San Francisco Liver Center Pilot/Feasibility Award (NIH grant P30DK026743). K.J. Evason is a Robert Black Fellow supported by the Damon Runyon Cancer Research Foundation (grant DRG-109-10). K. Asahina is supported by a grant from the NIH (R01AA020753). Our work on hepatic stellate cells and liver development was further supported by grants from the NIH (R01DK060322) and the Packard Foundation (to D.Y.R. Stainer).

Conflict of interest: The authors have declared that no conflict of interest exists.

Citation for this article:J Clin Invest. 2013;123(5):19021910. doi:10.1172/JCI66369.

Chunyue Yins present address is: Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Childrens Hospital Medical Center, Cincinnati, Ohio, USA.

Didier Y.R. Stainiers present address is: Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

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Hepatic stellate cells in liver development, regeneration, and cancer

In the first evidence of a natural intervention triggering stem cell-based regeneration of an organ or system, a study in the June 5 issue of the Cell Stem Cell shows that cycles of prolonged fasting not only protect against immune system damage a major side effect of chemotherapy but also induce immune system regeneration, shifting stem cells from a dormant state to a state of self-renewal.

In both mice and a Phase 1 human clinical trial involving patients receiving chemotherapy, long periods of not eating significantly lowered white blood cell counts. In mice, fasting cycles then flipped a regenerative switch, changing the signaling pathways for hematopoietic stem cells, which are responsible for the generation of blood and immune systems, the research showed.

The study has major implications for healthier aging, in which immune system decline contributes to increased susceptibility to disease as people age. By outlining how prolonged fasting cycles periods of no food for two to four days at a time over the course of six months kill older and damaged immune cells and generate new ones, the research also has implications for chemotherapy tolerance and for those with a wide range of immune system deficiencies, including autoimmunity disorders.

We could not predict that prolonged fasting would have such a remarkable effect in promoting stem cell-based regeneration of the hematopoietic system, said corresponding author Valter Longo, Edna M. Jones Professor of Gerontology and the Biological Sciences at the USC Davis School of Gerontologyand director of the USC Longevity Institute. Longo has a joint appointment at the USC Dornsife College of Letters, Arts and Sciences.

When you starve, the system tries to save energy, and one of the things it can do to save energy is to recycle a lot of the immune cells that are not needed, especially those that may be damaged, Longo said. What we started noticing in both our human work and animal work is that the white blood cell count goes down with prolonged fasting. Then when you re-feed, the blood cells come back. So we started thinking, well, where does it come from?

Prolonged fasting forces the body to use stores of glucose, fat and ketones, but it also breaks down a significant portion of white blood cells. Longo likens the effect to lightening a plane of excess cargo.

During each cycle of fasting, this depletion of white blood cells induces changes that trigger stem cell-based regeneration of new immune system cells. In particular, prolonged fasting reduced the enzyme PKA, an effect previously discovered by the Longo team to extend longevity in simple organisms and which has been linked in other research to the regulation of stem cell self-renewal and pluripotency that is, the potential for one cell to develop into many different cell types. Prolonged fasting also lowered levels of IGF-1, a growth-factor hormone that Longo and others have linked to aging, tumor progression and cancer risk.

PKA is the key gene that needs to shut down in order for these stem cells to switch into regenerative mode. It gives the OK for stem cells to go ahead and begin proliferating and rebuild the entire system, explained Longo, noting the potential of clinical applications that mimic the effects of prolonged fasting to rejuvenate the immune system. And the good news is that the body got rid of the parts of the system that might be damaged or old, the inefficient parts, during the fasting. Now, if you start with a system heavily damaged by chemotherapy or aging, fasting cycles can generate, literally, a new immune system.

Prolonged fasting also protected against toxicity in a pilot clinical trial in which a small group of patients fasted for a 72-hour period prior to chemotherapy, extending Longos influential past research.

While chemotherapy saves lives, it causes significant collateral damage to the immune system. The results of this study suggest that fasting may mitigate some of the harmful effects of chemotherapy, said co-author Tanya Dorff, assistant professor of clinical medicine at the USC Norris Comprehensive Cancer Center and Hospital. More clinical studies are needed, and any such dietary intervention should be undertaken only under the guidance of a physician.

We are investigating the possibility that these effects are applicable to many different systems and organs, not just the immune system, said Longo, whose lab is in the process of conducting further research on controlled dietary interventions and stem cell regeneration in both animal and clinical studies.

The study was supported by the National Institute of Aging of the National Institutes of Health (grant numbers AG20642, AG025135, P01AG34906). The clinical trial was supported by the V Foundation and the National Cancer Institute of the National Institutes of Health (P30CA014089).

Chia Wei-Cheng of USC Davis was first author of the study. Gregor Adams, Xiaoying Zhou and Ben Lam of the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC; Laura Perin and Stefano Da Sacco of the Saban Research Institute at Childrens Hospital Los Angeles; Min Wei of USC Davis; Mario Mirisola of the University of Palermo; Dorff and David Quinn of the Keck School of Medicine of USC; and John Kopchick of Ohio University were co-authors of the study.

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Fasting triggers stem cell regeneration of damaged, old immune system ...

Marc Howard was diagnosed with multiple myeloma. He will receive a transplant using his own cells later this year.

COLUMBUS, Ohio It all started with back pain that seemed to be progressively getting worse.

My back, the structure of my body, was like starting to deteriorate, and I could tell, he said. I'm tall, so when I started to lean over, and the pain and things of that nature, I'm like, yo, something's going on.

His longtime love Sonia Grant noticed, too. And she was right there to encourage him to get it checked.

When he did, doctors found holes in his spine where his bone had deteriorated. He had a vertebroplasty procedure to have those holes filled with bone cement. But that was not the end of his journey. In fact, it was really only the beginning.

After the surgery, he was okay for about a month, then I saw him (leaning) over again, and he couldnt get off the bed one day, Grant said. I said, uh uh, were going back up there (to the hospital). Theres something wrong.

And something was. Howard was diagnosed with multiple myeloma, a cancer that forms in the plasma cells.

I dont want to be the woe is me, Howard said. I want to be the success story for somebody, for the world to look at, like, that man went through a situation, and he made it.

And hes making it so far, with the help of Grant. Hes been doing weekly chemotherapy treatments and taking daily medication. Meanwhile, Grant is making sure hes eating his fruits and veggies and drinking plenty of water, too.

If youre not up to the challenge, I will help you get there, I will, Grant said. Because failure is just not a thing when it comes to fighting something like cancer. You gotta fight, you just gotta fight.

This fight will culminate with a major procedure later this fall via OhioHealths new Blood and Marrow Transplant Program. Howard will be one of the first patients to receive an autologous stem cell transplant, meaning the procedure will use his own cells.

Dr. Yvonne Efebera, the medical director for the program, explains this process is different than a procedure using donor cells.

BMT, blood and marrow transplant, is a process where, certain diseases require this, where non-functioning, deficient bone marrow or cancer cells are eliminated by giving high-dose chemotherapy, with or without radiation, and then replaced by new, healthy cells, Dr. Efebera said.

Shes been treating Howard throughout this process and points out that this is one of the benefits of the new program. Before, patients who needed transplants would have to be sent to other healthcare systems. Now, they can go from start to finish with the same clinical team.

Marc always wanted to be the first, she joked. Hes anxious to have his stem cells to be the first collected and the first admitted.

Both Howard and Grant are up to the challenge.

Its a battle, Grant said. Were halfway through the battle, and so, were going to get all the way to the end of the battle. Bruised, not broken. But were in the battle. But were going to get through it.

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Columbus man to be one of first to receive transplant via new OhioHealth program - 10TV

The board of directors of Grayson-Jockey Club Research Foundation has announced that it has authorized expenditure of $1,661,180 to fund15 new projects and 10 continuing projects at 16 universities as well as three career development awards. The 2022 slate of research brings Graysons totals since 1983 to more than $32.1 million to underwrite 412 projectsat 45 universities.

Grayson aims to support projects that address a wide range of equine health issues, and this diversity can be seen in our approved projects this year, said Jamie Haydon, president of the foundation. We are not able to fund these research projects and career development awards without the generosity of our donors, and we are grateful to them for recognizing the importance of equine veterinary research.

Below is an alphabetical list by school of the new projects:

Persistence of Antimicrobial Resistance in Horse FarmsLaura Huber,Auburn University

This project will determine the effect of antimicrobial pressure on multidrug resistant Rhodococcus equi. persistence in the soil of horse breeding farms in a 5-year period.

Evaluating extracellular vesicles from equine fetally-derived mesenchymal stem cells as an endometritis therapeuticFiona Hollinshead,Colorado State University

This project will beevaluating extracellular vesicles from equine fetally-derived mesenchymal stem cells as an endometritis therapeutic.

Development of a Palmar Osteochondral Disease ModelChris Kawcak,Colorado State University

The goal of this proposal is to develop an experimental model of palmar osteochondral disease in horses to better study disease progression and facilitate development of improved treatment strategies.

Development of a Vectored Vaccine to Equine Rotavirus AMariano Carossino, Louisiana State University

A novel viral vectored vaccine against equine rotavirus A (G3 and G14), the leading cause of foal diarrhea, will be designed and evaluated in mares and a neonatal mouse model as proof-of-concept.

Novel Strangles Vaccine Using CD40-Targeted DeliveryLuc Berghman,Texas A&M University

This project will be targeting bacterial components ofStreptococcus equispp. equito the horses immune surveillance cells (the APCs) that will result in a fast and strong immune response that will protect against strangles.

Trained Immunity in FoalsAngela Bordin, Texas A&M University

This project will study how giving oral live bacteria protects foals against infection byRhodococcus equi, the cause of severe and debilitating pneumonia in foals, for future development of a vaccine.

Immunogenicity in Foals of an mRNA Vaccine for R. EquiNoah Cohen, Texas A&M University

This study proposes to develop an mRNA vaccine delivered by inhalation to protect foals against pneumonia caused byRhodococcus equi.

Does Antibiotic Treatment Change the Microbial ResistomePaul Morley, Texas A&M University

This research will compare four antibiotic treatments to these protocols that can be selected to treat bacterial infections while also lessening the risks for promoting antibiotic resistance.

Immunomodulation and Exosomes to Enhance Tendon HealingSushmitha Durgam,The Ohio State University

This studyaims to characterize M1 and M2 macrophage-derived inflammatory factors and assess their impact on superficial digital flexor tendon tenocyte activities while examining the potential of extracellular vesicles/exosomes to enhance tendon healing.

Pharmacokinetics of Oral Mycophenolate Mofetil in HorsesGwendolen Lorch,The Ohio State University

This proposal will evaluate the pharmacokinetics of orally administered mycophenolate mofetil as a safe, effective, and inexpensive immunosuppressant drug for management of equine immune-mediated disease.

Equine Placentitis: New Approaches to an Old ProblemPouya Dini,University of California, Davis

The goal of this study is to identify pathogens involved in placentitis and investigate their interaction with the placenta using bioinformatics and in vitro studies to develop better diagnostic and treatment methods.

Motion of the Proximal Sesamoid Bones on Uneven FootingSusan Stover,University of California, Davis

This study proposes to determine how hoof conformation, shoeing, and uneven racetrack surfaces could contribute to fetlock breakdowns.

Influence of Vitamin D and Cortisol inR. Equi InfectionKelsey Hart,University of Georgia

This study will investigate how blood levels of cortisol and vitamin D are related to the development and progression ofRhodococcus equipneumonia in foals after natural exposure.

Fentanyl Matrix Patches in HorsesRachel Reed,University of Georgia

This study aims to show that fentanyl administered via patches placed on the skin is well-absorbed and represents a promising means of providing clinically relevant continuous pain relief to horses.

Sirolimus for the Control of Insulin DysregulationAndrew Van Eps,University of Pennsylvania

This study will evaluate the drug sirolimus (a potent suppressor of insulin production) for the treatment of insulin dysregulation (the most important cause of laminitis) in horses.

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Grayson authorizes more than $1 million for ongoing and new equine research in 2022 - EQUUS Magazine

Genes Dev. 2015 Jun 15; 29(12): 12031217.

1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA;

2Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195, USA;

3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA;

3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

2Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195, USA;

3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA;

2Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195, USA;

3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA

Tissues with defined cellular hierarchies in development and homeostasis give rise to tumors with cellular hierarchies, suggesting that tumors recapitulate specific tissues and mimic their origins. Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor and contains self-renewing, tumorigenic cancer stem cells (CSCs) that contribute to tumor initiation and therapeutic resistance. As normal stem and progenitor cells participate in tissue development and repair, these developmental programs re-emerge in CSCs to support the development and progressive growth of tumors. Elucidation of the molecular mechanisms that govern CSCs has informed the development of novel targeted therapeutics for GBM and other brain cancers. CSCs are not self-autonomous units; rather, they function within an ecological system, both actively remodeling the microenvironment and receiving critical maintenance cues from their niches. To fulfill the future goal of developing novel therapies to collapse CSC dynamics, drawing parallels to other normal and pathological states that are highly interactive with their microenvironments and that use developmental signaling pathways will be beneficial.

Keywords: brain tumor, cancer stem cell, glioblastoma, glioma, stem cell, tumor-initiating cell

Development is a coordinated summation of the individual cellular dynamics that build an organ, and the programs responsible for this construction are generally preserved in stem cells for organ homeostasis and tissue repair. Tumors are complex systems that recapitulate the complexity of organs or tissues with dynamic regulation and constituent cellular populations during tumor initiation, maintenance, and progression (Hanahan and Weinberg 2011). While many scientists have sought to reduce the complexity of cancer to a one-dimensional processfor example, characterizing cancers solely based on geneticsmost advanced cancers unfortunately remain nearly as lethal since the declaration of the War on Cancer in 1971. Targeted therapeutics offer a transient benefit for some cancer types with driving mutations, but even these tumors will develop resistance to overcome initially effective therapies that poison driving oncogenes.

Glioblastoma (GBM; World Health Organization grade IV glioma) is the most prevalent and lethal primary intrinsic brain tumor (Stupp et al. 2009). Unlike other solid tumor cell types, GBM widely invades the surrounding brain but rarely metastasizes to other organs. While halting steps to fight GBM are being made using targeted therapies (e.g., bevacizumab) or immunotherapies, GBM therapy remains focused on achieving maximal surgical resection followed by concurrent radiation therapy with temozolomide (TMZ; an orally available methylation chemotherapy) and subsequent additional adjuvant TMZ therapy. Conventional treatment offers patients with GBM additional survival time with generally acceptable quality of life, but a cure is never achieved. GBM represents one of the most comprehensively genomically characterized cancer types, leading to recognition of groups of tumors defined by transcription profiles (proneural, neural, classical, and mesenchymal), genetics (mutations of isocitrate dehydrogenase 1 [IDH1]), and epigenetics (CpG island methylator phenotype [CIMP]) (Weller et al. 2013). Long-term survivors are often, but not exclusively, patients with tumors harboring mutations in IDH1, which likely represent a different disease than most GBMs. Beyond IDH1 mutations and a few other biomarkers (deletion of chromosomes 1p and 19q in oligodendrogliomas, methyl guanine methyltransferase [MGMT] promoter methylation, etc.), the accumulated genetic characterization of GBMs has failed to impact clinical practice, suggesting that other discovery paradigms should also be considered.

The brain, like other organs with clearly defined cellular hierarchies in development and homeostasis (e.g., blood, breast, skin, and colon), gives rise to tumors with defined cellular hierarchies, suggesting that cancer replicates ontogeny (Reya et al. 2001). Atop the apex of cellular hierarchies are stem cells, which have been assumed to be rare, quiescent, self-renewing cells, but several highly proliferative organs (e.g., the intestine and skin) contain at least two pools of stem cells: one quiescent, and the other highly proliferative (Barker et al. 2010). Stem cells generate transient amplifying cells, which in turn create lineage-restricted progeny that are eventually fated to become the terminally differentiated effector cells.

Neural stem and progenitor cell (NSPC) pools vary in location and control during development, suggesting that different cellular hierarchies may be co-opted by brain tumors (Gibson et al. 2010; Lottaz et al. 2010). Informed by techniques used to enrich and characterize NSPCs, several groups in parallel demonstrated that gliomas and other primary brain tumors contain self-renewing, tumorigenic cells (Ignatova et al. 2002; Hemmati et al. 2003; Singh et al. 2003, 2004; Galli et al. 2004). The nomenclature for these cells has been controversial (as discussed below), with the dominant choice being cancer stem cells (CSCs) or tumor-initiating cells (importantly, these terms are not identical, as a CSC designation is more restrictive but also more informative) (). Unlike normal brain organizationwhere the generation of differentiated progeny is stage-specific (neurons and then glia during development) and derived from rapidly dividing progenitor cells and quiescent, multipotent stem cells that persist into adulthood and become activated upon differentiation (Rowitch and Kriegstein 2010)these populations have yet to be delineated in brain tumors. The ability to distinguish between self-renewing cells with stem and progenitor cell cycle properties and transcriptional signatures is likely to provide clarity with respect to nomenclature and the functional interplay between cells at the apex of the hierarchy. The challenges distinguishing CSCs from their progeny are derived, in part, from the limited recognition of points of relative stability (attractor states) in the landscape of cellular identity that define the stem cell state and transitions into (dedifferentiation) and out of (differentiation) a stable state (Chang et al. 2008). Much like the Heisenberg uncertainty principle in physics, our ability to observe the state of a cell is limited by our act of measurement. The presence of subatomic particles is confirmed in retrospect; similarly, the functional definition of both normal and neoplastic stem cells requires retrospective confirmation. The ability to prospectively distinguish glioma stem cells, which reside at the apex of tumor hierarchies, from their differentiated progeny remains challenging; however, stem cell biology faces a similar difficulty with normal stem cell identification. Of note, the CSC hypothesis does not claim a stem cell as the cell of origin for cancers, suggesting that CSCs do not need to adhere to all of the observed features of normal stem cells.

Definitions and functional characteristics of CSCs, tumor-initiating cells, and tumor-propagating cells

In the following sections, we provide an update on intrinsic and extrinsic regulators of the CSC state in GBM and discuss how the integration of genetics, epigenetics, and metabolism has shaped our understanding of how CSCs function to drive GBM growth. We also highlight future opportunities to further understand the complexity of CSC regulation through interaction with other cells (including immune cells) and how the translation of CSC-based therapies needs to take into account the cellular dynamics of CSCs, which rely on developmental signaling programs.

The heterogeneity of tumor cells has long been appreciated, but two decades ago, seminal work from Dick's laboratory (Bonnet and Dick 1997) described the isolation of a leukemia-initiating cell, the first purification of cancer stem-like cells, a population of cells that had originally been proposed to exist >150 years earlier (Sell 2004). The first prospective isolation of human NSPCs was performed using CD133 (Uchida et al. 2000) and prompted a search for brain tumor cells that shared the characteristics of NSPCs. A burst of studies soon followed describing brain CSCs in anaplastic astrocytoma (Ignatova et al. 2002), medulloblastoma, pilocytic astrocytoma, ependymoma, ganglioglioma (Hemmati et al. 2003; Singh et al. 2003), and GBM (Ignatova et al. 2002; Hemmati et al. 2003; Galli et al. 2004). Brain CSCs have subsequently been shown to be resistant to standard-of-care chemotherapy (Chen et al. 2012) and radiotherapy (Bao et al. 2006a), underscoring their role in disease progression and recurrence.

While cellular heterogeneity within CNS tumors is well recognized (Bonavia et al. 2011; Meacham and Morrison 2013), the nomenclature used to describe the self-renewing population of tumor cells with enhanced tumorigenic properties is far from uniform (see ). To date, many terms have been used to describe this population, including cancer/tumor/glioma/brain tumor stem cell, stem-like tumor cell, cancer-/tumor-/glioma-/brain tumor-initiating cell, and cancer-/tumor-/glioma-/brain tumor-propagating cell. This lack of uniformity has generated confusion and controversy by redirecting the focus away from the biology of these cells and their contribution to tumorigenic processes toward identifying markers that the cells express and whether tumor cells can be propagated as free-floating spheres. In addition, while the term stem cell is used, it does not necessarily mean these cells have been generated from a transformed stem cell, as there is evidence that multiple cell typesranging from stem cell to differentiated progeny, depending on the modelare amenable to oncogenic transformation. Therefore, in the current context, it is essential that the strictest functional assays continue to be performed and a singular term be used for studies using models that fulfill this criteria. As the accepted functional definition of a stem cell is the ability to self-renew and generate differentiated progeny, any claims for a CSC population must also demonstrate this capacity (). For brain tumors, this means the ability to generate a tumor upon intracranial transplantation that recapitulates the cellular heterogeneity present in the parental tumor. Unlike the designation of a tumor-initiating cell, CSCs cannot be investigated in isolation due to the required comparison with differentiated progeny. Prospective enrichment and depletion of tumorigenic and nontumorigenic cells demonstrate the presence of a cellular hierarchy. Cells that meet these criteria (tumorigenic and containing a cellular hierarchy) should be referred to as CSCs (or alternatives such as glioma stem cells, glioma CSCs, or brain tumor stem cells in the context of GBM). While the ability to grow as spheres is also evident in CSCs, it is not by default the defining feature of a self-renewing population of cells. In fact, the majority of spheres derived from both normal and neoplastic brain cells come from progenitor cells with limited self-renewal potential, not stem cells (Pastrana et al. 2011). Furthermore, high-passage cell lines, which do not offer the ability to accurately represent tumor complexity in vivo (Lee et al. 2006), should not be replaced with cells grown in long-term passage as spheres but rather with functionally validated CSC models, as this offers the best opportunity to more deeply model the complexity of brain tumors. Thus, although culture of glioma cells as neurospheres may not be required to maintain stemness (Pollard et al. 2009; Cheng et al. 2012), the microenvironment, including medium composition and culture conditions, does necessarily affect the characteristics of CSCs (Pastrana et al. 2011).

Functional criteria of CSCs. CSCs are defined by functional characteristics that include sustained self-renewal, persistent proliferation, and tumor initiation upon secondary transplantation, which is the definitive functional CSC assay. CSCs also share features with somatic stem cells, including frequency within a tissue (or tumor), stem cell marker expression (examples relevant to GBM and the brain are provided), and the ability to generate progeny of multiple lineages.

Great energy and passion have been devoted to the discovery, validation, and use of CSC enrichment methods. Demonstration of a cellular hierarchy demands methods to separate populations that can be functionally studied. Ideally, an enrichment method would be based on a property that defines an essential CSC feature (self-renewal, tumor initiation, etc.) that is immediately lost upon differentiation (i.e., a digital readout) and is usable with live cells. Currently, no such system exists for any cell type (normal or neoplastic) because biologic systems rarely exhibit all or none phenomena. Critics of the CSC hypothesis have held this limitation up as proof against CSCs; while the same limitations exist for even the best-characterized normal stem cell (hematopoietic stem cell), no scientists deny the existence of hematopoietic stem cells. Leukemia stem cells are considered a definitive tumor population, yet no marker signature for these cells is definitive (Eppert et al. 2011). A more sophisticated and nuanced use of enrichment systems that is informed by recognition of the diversity of GBMs can lead to context-specific methods to produce matched tumorigenic and nontumorigenic populations.

Most glioma CSC markers have been appropriated from normal stem cells, but the linkage between glioma CSCs and normal stem cells remains controversial. Many of the transcription factors or structural proteins essential for normal NSPC function also mark glioma CSCs, including SOX2 (Hemmati et al. 2003), NANOG (Ben-Porath et al. 2008; Suva et al. 2014), OLIG2 (Ligon et al. 2007), MYC (Kim et al. 2010), MUSASHI1 (Hemmati et al. 2003), BMI1 (Hemmati et al. 2003), NESTIN (Tunici et al. 2004), and inhibitor of differentiation protein 1 (ID1) (Anido et al. 2010). However, because of the limited utility of intracellular proteins for enriching CSCs from nonstem tumor cells (NSTCs) using traditional methods such as flow cytometry, a multitude of potential cell surface markers have been suggested, including CD133 (Hemmati et al. 2003), CD15 (also called Lewis x and SSEA-1 [stage-specific embryonic antigen 1]) (Son et al. 2009), integrin 6 (Lathia et al. 2010), CD44 (Liu et al. 2006), L1CAM (Bao et al. 2008), and A2B5 (Ogden et al. 2008). These types of cell surface markers mediate interactions between cells and the microenvironment, but dissociation of cells from their surroundings rapidly degrades the informational content of markers, requiring rapid utilization.

The first proposed marker, CD133 (Prominin-1), a cell surface glycoprotein expressed on neural stem cells, enriches for cells with higher rates of self-renewal and proliferation and increased differentiation ability (Singh et al. 2003). However, CD133 expression, rather than the AC133 surface epitope, should be used with care to enrich for any cells: Surface CD133 marks stem cells and decreases with differentiation, but the expression of Prominin-1 mRNA is not regulated with stemness (Kemper et al. 2010), suggesting that only the glycosylated surface protein CD133 is CSC-specific. The AC133 antigen marks the glycosylated molecule localized in lipid rafts that signals through PI3K and other key pathways to mediate interactions between a cell and its microenvironment (Wei et al. 2013). Most studies fail to recognize this role and use CD133 as a marker in cells that have been extensively cultured out of their microenvironment. Furthermore, the information contained in CD133 is context-dependent. CD133 mRNA, protein lysates, immunofluorescence, and FACS analysis for the AC133 glycoprotein have very different relationships to cell biology. Unfortunately, the complexity of these biomarkers has led to a reductionist view that has challenged the field due to the lack of consistency in methodology and models. It is nearly certain that CD133 is not universally informative in all tumors and has a false-negative rate for identifying CSCs (CD133-negative cells can be tumor-propagating in some tumors) (Beier et al. 2007). Additionally, the use of CD133 as a stem marker is complicated by the observation that expression of CD133 can be regulated at the level of the cell cycle, with potentially slow-cycling NSPCs lacking CD133 expression during G0/G1 cell cycle phase but still maintaining multipotency (Sun et al. 2009).

Although CD133 continues to be the most commonly used cell surface marker, other markers, such as integrin 6, have been proposed to segregate CSCs and NSTCs (Lathia et al. 2010). CD15/SSEA-1 and CD44 have also been proposed as possible markers, potentially with an association with specific subgroups of GBM (Bhat et al. 2013). These markers have utility but must be approached with caution. Each can mark a large percentage of cells, consistent with a high false-positive rate. Due to the current limitations in the functional assays defining CSCs, false-positive markers are sometimes claimed to be superior to functional identification, but markers lack significant utility in discovery studies, which benefit from greater specificity. Additionally, it is likely that no marker will ever be uniformly informative for CSCs because most tissue types contain multiple populations of stem cells expressing different markers and due to the inherent adaptability of cancer cells.

Several methods other than marker expression have been used to enrich for glioma CSCs, such as the abilities to grow as neurospheres in serum-free medium or efflux fluorescent dyes (Goodell et al. 1996; Kondo et al. 2004). Many investigators have used neurosphere culture to select for progenitor cells in the normal and neoplastic brain cells, but there are challenges with this approach. Neurosphere culture selects for a small fraction of the original tumor cells with bias toward progenitor features and expression of epidermal growth factor receptor (EGFR) and FGFR based on growth factors (EGF and FGF) added to the medium (Pastrana et al. 2011). This selection process eliminates the ability to prospectively enrich and deplete stem-like cells, preventing the delineation of a cellular hierarchy required to prove the presence of CSCs. Neurosphere culture selects for cells that can grow in stem cell medium; however, the selection of CSCs simply based on culture methods fails to recapitulate the heterogeneity of the original tumor in vivo as assessed by histological morphology, differentiated cell lineage, and gene expression (Lee et al. 2006; Lathia et al. 2011; Venere et al. 2011), a characteristic that CSCs acutely isolated using marker expression maintain (Singh et al. 2004). An alternative approach to CSC enrichment is the use of flow cytometry to isolate a side population containing CSCs, which is based on the hypothesis that stem cells contain drug efflux transporters (Yu et al. 2008). While this approach has identified a population of self-renewing cells in a mouse glioma model (Bleau et al. 2009), it has not been used successfully to enrich for self-renewing cells in human GBM (Broadley et al. 2011; Golebiewska et al. 2013), highlighting the model- and species-specific challenges of enrichment methods.

CSC markers, although useful to enrich populations of stem cells from nonstem cells, are not sufficient to define either population due to the lack of definitive markers. Functional validationthe observation of differences in stem cell characteristics of CSCs and NSTCsis essential to ensure that the enriched cells truly exhibit the functional characteristics of stem cells (). Various methods, both in vitro and in vivo, are employed to assess stem cell characteristics (self-renewal, proliferation, and ability to reproduce the complexity of the original tumor) of enriched cells. In vitro neurosphere formation assays test for both proliferation and self-renewal but fail to address cellular hierarchy and do not recapitulate the tumor microenvironment. Sphere formation is a surrogate of self-renewal capability andwhen performed in a limiting dilution formatstem cell frequency, but in vivo tumor formation assays are essential to claim the presence of CSCs.

The gold standard for CSC determination remains the ability of a limiting dilution of cells to recapitulate the complexity of the original patient tumor when transplanted orthotopically. The ability to derive heterogeneity is essential because populations of transit-amplifying cells may form a tumor but will only give rise to cells from their specific lineage. Heterotypic transplantation of cellsfor example, into the flank of the animalmay also be informative, but this technique lacks the proper microenvironmental cues of orthotopic implantation.

Glioma CSCs are regulated by six main mechanisms, which include intrinsic factors such as genetics, epigenetics, and metabolism as well as extrinsic qualities of niche factors, cellular microenvironment, and the host immune system (). The following sections describe the key features of each of these factors and highlight new advances in the topics of epigenetics mapping, single-cell heterogeneity, metabolism, and immunotherapy.

Regulation of CSCs. Cell-autonomous (intrinsic) and external (extrinsic) forces regulate the CSC state. Key intrinsic regulators include genetic, epigenetic, and metabolic regulation, while extrinsic regulators include interaction with the microenvironment, including niche factors and the immune system.

Through advances in genomic technologies, we now have a comprehensive picture of the genetic mutations and structural variants present in GBM (Atlas 2008; Brennan et al. 2013). Some of the most recurrent alterations include EGFR, IDH1, PDGFRA, HDM2, PIK3CA, and TERT promoter and PI3KR1 gain-of-function mutations or amplifications and mutations or deletions of the tumor suppressors PTEN, TP53, CDKN2A, NF1, ATRX, and RB1. While many of these mutations are prevalent in several other cancer genomes, several mutations are highly enriched in GBM, such as IDH1 mutations, which lead to a CIMP (G-CIMP) (Noushmehr et al. 2010). These studies highlight the significant degree of intertumoral heterogeneity present in GBM, which is further captured at both the transcriptional and epigenetic levels (Phillips et al. 2006; Verhaak et al. 2010), and also underscore the complexity of the clonal evolution and clonal diversity that occur during the genesis of GBM and their bearing on the shape and structure of the CSC hierarchy. While both genetic and epigenetic landscapes define functionally distinct clones during tumor evolution, epigenetic differences likely account for the functional differences between cells within the hierarchy.

Epigenetic maintenance of the CSC state is regulated largely at the level of transcriptional and chromatin regulation. CSC regulation converges on MYC, which occurs in the presence of MYC-mediated cancer cell survival and proliferation programs (Wang et al. 2008; Zheng et al. 2008; Wurdak et al. 2010; Chan et al. 2012; Fang et al. 2014). Additional transcription factors have been identified as important for CSC identity, including STAT3 (Sherry et al. 2009), SOX2 (Gangemi et al. 2009), FOXM1 (Joshi et al. 2013), FOXG1 (Verginelli et al. 2013), GLI1 (Clement et al. 2007), ASCL1 (Rheinbay et al. 2013), ZFX (Fang et al. 2014), NANOG (Zbinden et al. 2010), and ZFHX4 (Chudnovsky et al. 2014), which recruit necessary chromatin remodeling factors to promote maintenance of the glioma CSC state. By using epigenome-wide mapping of cellular chromatin state, Suva et al. (2014) identified a core set of four transcription factors in proneural GBM able to reprogram differentiated tumor cells into glioma CSCs. These investigators showed that POU3F2, SOX2, SALL2, and OLIG2 are master transcription factors required to maintain the tumor-forming capability of these cells, suggesting that mediators of stem cell programs could capture the oncogenic capacity of CSCs. In addition to transcription factors, regulators of nucleosome structure have also been reported to maintain the CSC state. The mixed lineage leukemia 1 (MLL1) protein has been shown to maintain the CSC phenotype through activation of HOXA10, which subsequently regulates a network of homeobox genes that is required for tumor maintenance (Heddleston et al. 2012; Gallo et al. 2013). Similarly, the H3K27 methylase EZH2 has been shown to be important for CSC maintenance through its function as a regulator of both Polycomb-repressive domains and STAT3 signaling (Kim et al. 2013). The BMI1 Polycomb ring finger oncogene regulates both normal neural stem cells and GBM cells (Bruggeman et al. 2007).

These studies highlight the importance of understanding the dynamics of core transcription factors in maintaining stem cell state and the effect that these factors have on shaping the chromatin landscape of cells within the tumor hierarchy.

Single-cell RNA sequencing (RNA-seq) interrogation of cellular heterogeneity within GBMs identified novel genes predominantly present in GBM CSCs compared with differentiated cells and provocatively detected cells of multiple GBM subtypes within single tumors, drawing into question the utility of subtyping tumors and targeting specific subtypes (Patel et al. 2014). Furthermore, these investigators described an inverse correlation between stem signature and cell cycle gene expression, suggesting that the cells that form neurospheres in culture cycle more slowly compared with differentiated and differentiating tumor cells. A parallel, single-cell functional analysis of GBMs confirmed a strong variation of genomics and response to therapy (Meyer et al. 2015). Additional detailed analysis of heterogeneity of this type will greatly expand our understanding of the differences between tumor cells both within and among GBM patients and improve the characterization of glioma CSCs.

GBM CSCs reside in varied tumor microenvironments that limit nutrients, such as glucose and oxygen. Under such conditions, cancer cells, including glioma CSCs, exhibit the Warburg effect, a metabolic shift toward aerobic glycolysis and the accumulation of lactate in exchange for sustained ATP production and metabolite generation for macromolecule synthesis. Glioma CSCs demonstrate plasticity in the metabolic pathways used in response to metabolic restrictions and may shift toward the use of the pentose phosphate shunt (Vlashi et al. 2011; Kathagen et al. 2013). This inherent persistence of CSCs under hypoxic and acidic conditions as well as the preferential utilization of HIF-2 signaling compared with NSTCs and normal progenitors promote the maintenance of self-renewal, proliferation, and survival (Li et al. 2009b). Similarly, in conditions of nutrient deprivation such as low glucose, glioma CSCs outcompete neighboring NSTCs for glucose uptake through preferential up-regulation of the high-affinity GLUT3 transporter (Flavahan et al. 2013). A consequence of altered metabolic state is the production of reactive oxygen species. Glioma CSCs not only are dependent on NOS2 activity for promoting tumor growth but also synthesize nitric oxide through the specific up-regulation of NOS2 protein (Eyler et al. 2011). Importantly, in GBM, cellular metabolic characteristics are often genetically hardwired, such as recurrent IDH1 mutations, which are commonly observed in proneural GBM. Mutant IDH1 leads to a gain-of-function enzymatic activity, causing accumulation of 2-hydroxyglutarate, an oncometabolite that inhibits the TET1 and TET2 demethylases to cause aberrant hypermethylation of DNA and histones. While the function of IDH1 mutations in the context of CSCs is not directly defined, IDH1 mutations induce a loss of differentiation, preventing the terminal differentiation of lineage-specific progenitors (Lu et al. 2012). Moving forward, integrated metabolomic and epigenomic profiling may reveal other examples of intricate relationships between metabolism and epigenetic programs and their influence on the glioma CSC state.

Brain development is orchestrated by a series of regulatory pathways with spatially and temporally controlled activity. Notch and NF-B (nuclear factor B) signaling instructs the fate of NSPCs, with the guidance and lineage commitment of progeny dictated by pathways that include the ephrins and bone morphogenetic proteins (BMPs). In a manner that mimics aberrant differentiation, CSCs co-opt developmental programs to maintain an undifferentiated state, increasing their survival and maintenance. Common pathways activated in CSCs include Notch, BMP, NF-B, and Wnt signaling (Li et al. 2009a; Day et al. 2013; Rheinbay et al. 2013; Lubanska et al. 2014; Yan et al. 2014). Collectively, niche factors represent an overriding theme in CSC biology, where stem and progenitor cell features provide selective advantages to maintain tumor growth (). These pathways may be activated through a combination of genetic and epigenetic alterations in addition to microenvironmental and metabolic factors.

The Notch pathway plays a role during neural development, functioning to inhibit neuronal differentiation and sustain NSPC populations. This pathway is co-opted in GBM, where aberrant NOTCH activation stimulates astrocytes to assume a stem-like state accompanied by increased proliferation (Jeon et al. 2008). The importance of Notch signaling in glioma CSC biology is highlighted by the convergence on this pathway from other pathways and exogenous factors, such as hypoxia, eNOS signaling, and response to radiation (Charles et al. 2010; Wang et al. 2010; Qiang et al. 2012). The dependence of glioma CSCs on Notch signaling is further supported by experiments demonstrating depletion of CSCs by treatment with -secretase inhibitors (Fan et al. 2006, 2010).

As BMPs direct NSPC fate toward an astroglial lineage, these signals have been proposed as a possible differentiation therapy for GBM (Piccirillo et al. 2006). Despite the presence of BMP expression in primary GBM tissue, glioma CSCs are highly resistant to the differentiation effects of BMPs in a process that occurs through at least two distinct cell-autonomous mechanisms: the shift to a fetal BMP receptor expression in glioma CSCs through recruitment of the transcriptional repressor EZH2 (Lee et al. 2008) and the secretion of BMP antagonists, specifically Gremlin1, by CSCs to protect against endogenous BMP-mediated differentiation (Yan et al. 2014). In this manner, CSCs generate differentiated progeny that provide supportive cues to the parental cells (e.g., Notch ligands, interleukin-6 [IL-6], and extracellular matrix) while resisting differentiation signals.

The NF-B pathway has emerged as an important regulator of GBM cell survival and identity through an endogenous cell stress response transcriptional program (Bhat et al. 2013). The A20 protein (TNFAIP3), a mediator of cell survival and the NF-B pathway, is overexpressed in CSCs compared with NSTCs (Hjelmeland et al. 2010). Supporting these findings, Sema3C and its receptors, PlexinA2 and PlexinD1, are also coordinately expressed in CSCs and activate Rac1 and NF-B in an autocrine/paracrine loop to promote CSC survival (Man et al. 2014).

GBM CSCs have also been shown to be highly dependent on Ephrin receptor signaling for survival and the maintenance of stem cell properties. Specifically, Ephrin A molecules and the EPHA2 and EPHA3 receptors are highly expressed in glioma CSCs and potentially function through the negative regulation of mitogen-activated protein kinase (MAPK) signaling (Binda et al. 2012; Day et al. 2013).

Wnt signaling is highly active in CSCs and is critical for the maintenance of the stem cell phenotype. An integrated genomic and biological analysis identified PLAGL2 as highly amplified in gliomas with functional suppression of CSC differentiation through modulation of Wnt/-catenin signaling (Zheng et al. 2010). Comprehensive mapping of chromatin modifications in CSCs and their NSTC counterparts revealed widespread activation of Wnt pathway genes through loss of Polycomb-mediated repression. The CSC chromatin landscape is thought to be dependent on achaete scute family basic helixloophelix (bHLH) transcription factor 1 (ASCL1), which activates Wnt signaling through negative regulation of dickkopf WNT signaling pathway inhibitor 1 (DKK1) (Rheinbay et al. 2013). Hedgehog signaling in the CNS is mediated in part by NSPC communication with the cerebrovascular fluid through primary cilia. Gliomas contain primary cilia, and the resulting CSCs are dependent on hedgehog signaling (Bar et al. 2007; Clement et al. 2007; Ehtesham et al. 2007).

Given the role of growth factors in normal brain development, it is not unexpected that numerous canonical growth factor signaling pathways have been shown to contribute to GBM maintenance and function. PDGFR signaling promotes CSC survival, self-renewal, and invasion and tumor growth through downstream STAT3 activation (Kim et al. 2012). Similarly, glioma CSCs preferentially express the IL-6 receptor, which also promotes convergent signaling upon STAT3 activation (Wang et al. 2009).

EGFR signaling has also been reported to contribute to CSC maintenance through the activation of AKT, the recruitment of SMAD5, and the induction of ID3, IL-6, and IL-8. This suggests a potential hypothesis in which the EGFR and PDGFR pathways are linked by IL-6 signaling. A potential alternate hypothesis is the presence of distinct CSC populations dependent on different growth factor receptor signaling pathways. Supporting this latter notion, EGFR inhibition promotes expansion of a cMET growth factor receptor-positive population of CSCs (Jun et al. 2014). Furthermore, elevated cMET expression is important for CSC maintenance, tumorigenicity, and resistance to radiation (Joo et al. 2012).

Aligned with its role in stress responses, transforming growth factor (TGF-) stimulates CSC self-renewal. Autocrine TGF- signaling permits retention of stemness through positive regulation of SOX2 and SOX4 expression (Ikushima et al. 2009). A distinct subset of TGF--dependent CSCs expresses CD44 and ID1 (Anido et al. 2010), which are markers of functionally distinct CSCs. A crucial mediator of the TGF- response in CSCs is the BMI1 protein, which connects stem cell programs and ER stress pathways through the transcriptional repressor ATF3 (Gargiulo et al. 2013).

Immune suppression is a hallmark of cancer (Hanahan and Weinberg 2011); while the brain possesses a unique series of immune surveillance mechanisms that become active during pathogenic states (Ransohoff and Engelhardt 2012), brain tumors have been characterized as immunosuppressive (Platten et al. 2001; Fecci et al. 2006). There is increasing enthusiasm for immunotherapy strategies based on the limited success of signaling pathway inhibitors and anti-angiogenic agents in brain tumors and the success of immunotherapy in melanoma. Immunotherapies for brain tumors include cellular (adoptive T-cell transfer and chimeric antigen receptor engineered T cells), vaccination, and immunomodulatory therapies targeting immune checkpoints (including anti-programmed death 1 [PD1], PD ligand 1 [PD-L1], and cytotoxic T lymphocyte-associated protein 4 [CTLA4] antibodies) (Reardon et al. 2014). Reversing tumor-induced immune suppression by increasing cytotoxic cell function and reducing suppressor cell function may unleash the endogenous immune response. Immunologic therapies may offer an additional benefit, as most strategies do not require intracranial delivery, a major restriction point for many oncologic treatments. While CSCs are key drivers of tumor growth, CSC interactions with the immune system and potential exploitation in immunotherapy are under active investigation (). These studies will require innovative approaches, as the majority of CSC studies involve xenograft models that lack major immune cell components, and many mouse models have reduced cellular heterogeneity. However, the information obtained from mouse model approaches is likely to be informative for the human immune response, as genetically engineered mouse models can recapitulate key aspects of brain tumor immunosuppression (Kong et al. 2010).

Proposed features of CSCs. Non-cell-autonomous aspects of CSCs may drive tumor growth but also serve as points of fragility. These include the increased ability to invade through the brain parenchyma, immune evasion, relationship with a niche, and promotion of angiogenesis.

Despite these challenges, there is building evidence that CSCs directly modulate the immune system. In coculture studies, CSCs induced regulatory T cells while inhibiting proliferation and cytotoxic T-cell activation with a concomitant induction of cytotoxic T-cell apoptosis, mediated via PD1 and soluble galectin-3 (Di Tomaso et al. 2010; Wei et al. 2010). Other CSC-secreted factors include IL-10 and TGF-, which also suppresses tumor-associated microglia/macrophage function and generates a more immunosuppressive (M2) phenotype (Wu et al. 2010). Another immunotherapy approach that may benefit from CSC targeting is the development of anti-tumor vaccines. Current vaccine efforts have focused on tumor-specific antigens (such as EGFRvIII) or whole tumor cell lysates, and there is evidence from preclinical models that CSC lysates are more effective in generating dendritic cell (DC) vaccines than differentiated cells (Pellegatta et al. 2006; Xu et al. 2009). CSCs modulate T-cell and tumor-associated microglia/macrophage function through secreted factors (Zhou et al. 2015), which may be exploited in the development of vaccine strategies or in combination with other drugs (Sarkar et al. 2014). These data provide a rationale for future studies investigating how the interaction between CSCs and other immune cell populations may drive immune suppression and in vivo interrogations into how CSC targeting may alter the immune activation status. Evaluating changes in CSC populations as a result of immunotherapy will also be essential, as will be evaluating combinatorial targeting strategies using immunotherapies and anti-CSC approaches.

Most conventional anti-neoplastic therapies target proliferating cells, but the malignancy of advanced cancers also derives from effects on the immune system, vasculature, and invasion/metastasis (). GBMs infiltrate the surrounding brain, precluding curative surgical resection. Infiltrative tumors must adapt to new environments, including the formation of new vessels to obtain nutrients. GBMs express proangiogenic growth factors (Batchelor et al. 2007), with CSCs driving neoangiogenesis with high levels of VEGF (Bao et al. 2006b). The humanized monoclonal antibody bevacizumab was developed to target VEGF to inhibit angiogenesis and has been used to treat recurrent GBM (Cohen et al. 2009). Bevacizumab attenuates tumor size, but the surviving tumor may display increased invasion in human and mouse models (de Groot et al. 2010), potentially due to a release of c-MET inhibition (Lu et al. 2012). Cancer cells often activate redundant angiogenic pathways in response to VEGF pathway inhibition (Atlas 2008). CSCs located at the perivascular niche are in close contact with the endothelial cells (Calabrese et al. 2007), permitting engagement of endothelial cell Notch ligands with glioma CSC Notch receptors to activate Notch signaling, which supports self-renewal of glioma CSCs (Zhu et al. 2011). CSCs also contribute to vascular structure through transdifferentiation into pericytes to promote tumor growth (Cheng et al. 2013). Inhibition of CSC-derived pericytes disrupts angiogenesis and inhibits tumor growth, directing attention toward nonendothelial cell targeting strategies. Anti-angiogenic drugs in current use have failed to provide a significant survival benefit to GBM patients (Gilbert et al. 2014), suggesting that a benefit may exist to investigating the mechanisms by which tumor cells regulate angiogenesis and that contribute to tumor growth and maintenance to efficiently target the GBM vasculature.

The mainstay treatment of GBM involves surgery, concurrent radiation with chemotherapy, and adjuvant chemotherapy with TMZ (Stupp et al. 2009). Despite advances in the field, the overall survival rate remains only 1519 mo (Stupp et al. 2009). The high degree of tumor heterogeneity in GBM contributes to treatment failure, to which functional and molecular heterogeneity and aberrant receptor tyrosine kinase (RTK) activity all contribute. CSCs located at the top of the hierarchy initiate and maintain the tumor after treatment (Chen et al. 2012). Glioma CSCs have also been shown to contribute to radiation resistance by increasing the DNA damage response machinery (Bao et al. 2006a). In terms of molecular heterogeneity, different subtypes of GBM with distinct molecular profiles coexist within the same tumor and likely exhibit differential therapeutic responses (Sottoriva et al. 2013). For example, several RTKs, including PDGFR in the proneural and EGFR in the classical subtype, are altered in GBM (Verhaak et al. 2010). The abnormal activation of RTKs involves many pathways that are redundant and can initiate and maintain downstream signaling, making tumors refractory to treatment (Stommel et al. 2007). A recent single-cell analysis of primary GBM patients showed that cells from the same tumor have differential expression of genes involved in oncogenic signaling, proliferation, immune response, and hypoxia (Patel et al. 2014). Furthermore, an increase in tumor heterogeneity was associated with a decrease in patient survival. The addition of TMZ to radiation has increased median survival by several months (Stupp et al. 2009), but lineage tracing studies in mouse models demonstrate that CSCs repopulate brain tumors after TMZ treatment (Chen et al. 2012). A number of molecular mechanisms have been identified that mediate the therapeutic resistance of CSCs to cytotoxic therapies, including the DNA damage checkpoint, Notch, NF-B, EZH2, and PARP (Bao et al. 2006a; Wang et al. 2010; Bhat et al. 2013; Venere et al. 2014; Kim et al. 2015), which suggests that CSCs develop multiple mechanisms of resistance that may require combinations of targeted agents. Moving forward, these studies demonstrate the importance of understanding the molecular alterations that are present in recurrent tumors and how these influence the structure of cells within the tumor hierarchy. In addition, it is necessary to consider that therapeutic resistance mechanisms may not be solely innate but may evolve from exposure to microenvironmental factors such as hypoxia and acidic and metabolic stress (Heddleston et al. 2009; Li et al. 2009b; Hjelmeland et al. 2011; Flavahan et al. 2013; Xie et al. 2015).

Conventional treatment for GBM promotes a transient elimination of the tumor and is almost always followed by tumor recurrence, possibly with an increase in the percentage of CSCs (Auffinger et al. 2014), as CSCs are involved in tumor recurrence and therapeutic resistance (Bao et al. 2006a; Chen et al. 2012). To effectively eliminate CSCs, it is critical to target their essential functions and their interactions with the microenvironment. Treatment with TMZ may kill CSCs that contain higher expression of the DNA repair protein MGMT; however, TMZ cannot prevent self-renewal of CSCs that contain MGMT (Beier et al. 2008). Another feature of CSCs is their ability to evade apoptosis. A potential therapeutic strategy would be the use of PARP inhibitors to enhance apoptosis under genotoxic damage. When the PARP inhibitor ABT-888 was used in combination with TMZ and radiation in GBM cell lines, apoptosis increased, and cells were sensitized to therapy (Barazzuol et al. 2013). GBMs thrive in harsh microenvironments characterized by hypoxia and limited nutrient availability. The HIF family of transcription factors is involved in promoting angiogenesis and cell migration in hypoxic regions (Kaur et al. 2005), and several drugs have been developed to target this gene family, with a few undergoing clinical trials. For example, as described previously, glioma CSCs reprogram their metabolic machinery and preferentially take up glucose to survive in environments with limited nutrients by expressing the high-affinity glucose transporter GLUT3 (Flavahan et al. 2013). GLUT3 therefore represents a promising therapeutic target for potential selective inhibition of CSCs. Epigenetic modifications are manifest in tumor recurrence (Nagarajan and Costello 2009). Histone acetylation and methylation are reversible and can be targeted by drugs; the histone deacetylase (HDAC) inhibitor vorinostat is currently in clinical trials (Bezecny 2014). Immunotherapy is an additional emerging therapeutic approach for GBM. The development of vaccines based on heat-shock proteins, EGFRvIII (Del Vecchio et al. 2012), and DCs (Terasaki et al. 2011) has shown promising results in clinical trials. ICT-107, a patient-derived DC vaccine developed against six antigens highly expressed in glioma CSCs (Phuphanich et al. 2013), is currently under clinical evaluation for use in patients.

Some of the challenges of developing therapeutic targeting agents are derived from the lack of universally informative markers to identify CSCs and the common molecular pathways shared by CSCs and NSPCs. The understanding of the biology of the CSCs and how these cells interact with their microenvironment in combination with the genetic and epigenetic landscape in GBM will be essential to develop more effective therapies.

As biological observations have revealed increasing levels of complexity, mathematical modeling approaches have provided a framework to understand the dynamic complexity of stem cell self-renewal and differentiation. By use of proliferation data and lineage tracing analysis, quantitative models have been generated for tissue-specific stem cells that have provided insight into the kinetics of cell fate choice and tissue development (Blanpain and Simons 2013). Similar approaches have been taken to reduce the complexity of CSCs. A network-based model has suggested that CSCs can transition between plastic (proliferative, symmetrically dividing, and less invasive) and rigid (quiescent, asymmetrically dividing, and more invasive) networks that can be modulated by extrinsic stressors, such as hypoxia, inflammation, and therapies (Csermely et al. 2015). Testing this model with biological data is likely to provide additional insights into the complexity of CSCs and identify points of fragility for additional therapeutic development. Mathematical approaches have also been used to evaluate the dynamics of GBM growth. Proliferation and invasion are phenotypes that have been modeled (Harpold et al. 2007). By use of a model that takes into account rates of proliferation and invasion in combination with imaging data, it has been proposed that IDH1 mutant tumors are actually less proliferative and more invasive (Baldock et al. 2014). Clinically relevant parameters, such as identifying optimal radiation schedules, have also been modeled using genetically engineered mice (Leder et al. 2014). Additionally, quantitative approaches have been developed to model the events leading to intertumoral and intratumoral heterogeneity in both human patient specimens (Sottoriva et al. 2013) and mouse models (Cheng et al. 2012). Integrating mathematical approaches into future CSC studies will provide an opportunity to identify key pathways essential for self-renewal and will predict responses to therapeutic perturbations.

GBM provides an excellent system to investigate the importance of CSCs. While there is a standard set of assays used to enrich for and identify CSCs, it remains unclear whether multiple CSC populations exist in different niches (perivascular and hypoxic) and possess different characteristics (slow vs. rapid cycling) as well as how key developmental signaling pathways are used by each of these populations. In addition, while a hierarchy is in place for GBM, the current view of CSCs and NSTCs is mutually exclusive and lacks a progenitor cell population that serves as an intermediate for differentiated progeny generation from somatic stem cells. Mouse studies have revealed that multiple stem and progenitor cell populations have the capacity to give rise to tumors upon oncogenic transformation, but it remains unclear whether there is a single cell of origin for the human disease or, more likely, whether multiple cells of origin exist and how this may be linked to genetic diversity. Making inroads into these unresolved questions will refine the experimental foundation upon which translational studies aiming to develop novel anti-CSC therapies are built and provide key signaling pathways responsible for CSC maintenance that are amenable for targeting.

The extensive molecular characterization of gliomas of all grades has permitted the recognition that the continuum of tumor grade has hidden a set of genetically distinct diseases. IDH1 mutations produce an oncometabolite, 2-hydroxyglutarate, that reprograms cellular chromatin to assume a stem-like state (Lu et al. 2012). Thus, IDH1 mutant gliomas may have a relatively flat hierarchy, with most tumor cells acquiring stem-like features early in tumor initiation. In contrast, primary GBMs accumulate a greater diversity of genetic and epigenetic alterations, which is associated with a more vertical cellular hierarchy. This duality of tumor biology resembles that of the two forms of head and neck cancers. Human papilloma virus-induced head and neck cancers are morphologically uniform and, like IDH1 mutant gliomas, are more responsive to therapies. Alcohol- and tobacco-associated head and neck cancers harbor more mutations and display a worse outcome with a reliable cellular hierarchy. Large-scale genomic sequencing has informed commonalities among cancer types based on driving genetic lesions. It is possible that similar patterns will be appreciated with cancer types based on epigenetic and cellular hierarchies, creating broader opportunities to improve diagnostics and therapeutics. In fact, expanding the organizational structures is likely to be a useful approach to increase our understanding of complex disease states. Many diseases display heterogeneous aspects that are governed by both cell-autonomous and microenvironmental forces. With the success of immunotherapy approaches to activate the immune system via immune checkpoint inhibition in cancers such as melanoma, understanding how GBM and, in particular, CSCs interface with the immune system is an immediate priority. An alternative view of heterogeneity and therapeutic response may also be informative for future studies. For example, bacterial infections contain distinct populations of cells that have different proliferative potential and responses to therapy. Viable but nonculturable bacteria and latent infections, including tuberculosis, may be found in particular niches associated with inflammation, hypoxia, acidic and nitrosative stress, and nutrient restriction (Oliver 2010). Most antibiotics, like anti-neoplastic agents, are directed against the proliferative population, leaving a resistant population behind. Novel methods are being used to screen for new agents that target resistant bacteria, such as latent tuberculosis (Bryk et al. 2008). Nathan (2004) suggested that essentiality is conditional, and the conditions defining essentiality are multiple in the context of latent infections. An identical view can instruct CSC targeting efforts as we grow in our understanding of the governing stimuli both internal and external to CSCs.

One infrequently discussed point is a re-equilibration of a cellular hierarchy in tumors generated from CSCs. If cell-autonomous advantages were the sole determinant of the differentiation state of tumor cells, CSCs would represent the majority of tumor cells, as the evolutionary drive toward increased fitness would provide a selective advantage to CSCs. At steady state (in distinction from homeostasis), tissues balance competing requirements through multiple levels of interaction among stem cells, progenitor cells, and differentiated progeny. Collectively, the individual cellular dynamics in cancer permits tumors to respond to exogenous insults (cytotoxic therapies, immunologic attack, etc.) to maintain the aberrant organ system. These dynamics are also at play within the cellular hierarchy in which CSCs give rise to NSTCs, and, when necessary, NSTCs give rise to CSCs to maintain the cellular equilibrium necessary for optimal tumor growth. CSCs should not be considered a model to simplify the modeling of GBMs and other cancers, but rather the CSC hypothesis constitutes an additional level of complexity that contributes to the malignancy of cancers. As CSCs reside in multiple niches governed by different molecular programs, there will not be single anti-CSC targeted therapeutics with broad activity; instead, CSCs will demand multitargeted approaches. Patients with GBMs are in desperate need of improved therapies. The real validation of CSCs will come with better treatments due to the integration of CSCs into drug development.

We sincerely apologize to those investigators whose work we were unable to cite due to space limitations. We thank Amanda Mendelsohn (Center of Medical Art and Photography, Cleveland Clinic) for assistance with figure preparation. We also thank our funding sources: The National Institutes of Health (grants CA154130, CA171652, CA169117, NS087913, and NS089272 to J.N.R., and CA157948, CA191263, and NS083629 to J.D.L.); Sontag Foundation (J.D.L.); Research Programs Committees of Cleveland Clinic (J.N.R); and James S. McDonnell Foundation (J.N.R). S.C.M. is supported by a Canadian Institutes of Health Research Banting Fellowship. Work in the Lathia laboratory is also supported by the Lerner Research Institute, Case Comprehensive Cancer Center, Voices Against Brain Cancer, Blast GBM, the Ohio Cancer Research Associates, Research Scholar Award from the American Cancer Society, V Scholar Award from the V Foundation for Cancer Research, and grant IRG-91-022-18 to the Case Comprehensive Cancer Center from the American Cancer Society.

Original post:
Cancer stem cells in glioblastoma

First-responder cells launching the repair after a heart attack are so frantic about fixing the damage that they promote more inflammation than necessary, new research in mice suggests.

Based on those findings, scientists are pursuing interventions that would bring more balance to the healing process after a heart attack.

In a series of studies, the researchers have identified the cellular events that lead to a call for reinforcements an extra wave of the first responders to the site of repair. This process leads to the release of proinflammatory proteins at a point when they arent needed, creating conditions that may threaten optimum healing of the heart.

The first-responder cells in question are neutrophils, the most abundant of all white blood cells whose job is to heal wounds and clear away infection. Researchers are exploring potential drugs or genetic techniques that could block the call for neutrophil backups or limit the release of proteins that drive up inflammation.

We just want to prevent further damage that happens to the heart by toning down the neutrophil response, said Prabhakara Nagareddy, associate professor of cardiac surgery in The Ohio State University College of Medicine. The neutrophils are misguided and they overreact. How can we tame them? How can we bring that down?

We started looking at the role of inflammation in scar formation to see if we could potentially alter that process.

The most recent study on this work is published in the Jan. 4, 2022, issue of the journal Circulation.

The loss of nutrients and oxygen during a heart attack causes death of cardiomyocytes (cardiac muscle cells) and other cells that eventually result in scar formation at the site of that loss. Even with restoration of blood flow to the damaged part of the heart, scarring cant be completely avoided.

You have an injury, and the body will take care of the injury. Sometimes not doing anything is fine because the body has a healing process, Nagareddy said. But medicine is all about healing better and identifying the mechanism that improves healing.

For these studies, the researchers induce heart attack symptoms in mice and use those models to observe how inflammation starts and increases during the heart repair process.

Neutrophils are definitely a key part of the problem. In an earlier study, Nagareddy and colleagues found that heart-attack patients with higher numbers of neutrophils in their blood upon hospital admission, or even after doctors restored blood flow, had the worst outcomes.

However, because neutrophils are vital to all wound healing and infection fighting, their first-responder role in heart repair cannot be bluntly targeted for elimination. Instead, the team has zeroed in on signals sent to the immune response control center the bone marrow that trigger ramped-up production of neutrophils.

As part of that investigation, the researchers found that the first wave of neutrophils to arrive at the damaged heart consider the injury so severe that they sacrifice themselves to prevent further damage, releasing their entire contents including proteins called alarmins. These alarmins in turn activate sensors in a second wave of neutrophils, priming those cells for more intense action.

These primed neutrophils then do something unexpected: They reverse migrate from the heart to the bone marrow and release a proinflammatory protein there, which prompts stem cells in the bone marrow to churn out even more neutrophils all processes that perpetuate inflammation at a time when its no longer needed for heart repair.

In the most recent paper, experiments in mice using genetic techniques or drugs uncovered at least two potential targets to consider for intervention: limiting the primed neutrophils reverse migration or suppressing neutrophils release of the proinflammatory protein in the bone marrow. The studies showed that successful inhibition of either mechanism led to better cardiac outcomes and less scarring in the mice.

Neutrophils dont see the difference between one tissue and another, so we need to focus on a signaling pathway or mechanism while the neutrophils are busy, and find the right time to intervene, Nagareddy said. My lab is hoping to find anti-inflammatory therapies that could be administered just before arteries are unclogged. It would have to be administered while the inflammatory response is in progress we need to have a certain amount of inflammation in the heart to begin the repair mechanisms.

This work was supported by grants from the National Institutes of Health and startup funds from Ohio State.

Ohio State co-authors on the most recent Circulation paper include Gopalkrishna Sreejit, Robert Jaggers, Baskaran Athmanathan, Ki Ho Park, Jillian Johnson, Albert Dahdah and Jianjie Ma.

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First-responder cells after heart attack prompt ...

I get sent stuff by patients all the time. This week a patient reached out about Riverside Biologic Healths IV and injection at home umbilical cord stem cell service. Hence, lets dig in.

I received two unsolicited emails from a patient describing himself as an advocate for Regenexx:

High Tina, please pass on my request to dr. Centeno about the back ground of Riverside biologics, and also the director Dr. Neil Riodan. I attended one of their sales meetings and listened to some fantastic claims of which most were lies. One item of interest I did not understand was that the DNA becomes inactive when the cells are extracted from the fetus because of the HLA protein so there is no possibility of rejection. I tried to research this but did not get a clear understanding. All the other claims were of course the Wild West. The marketing was interesting in that in my state on rep would send post cards in each city or town inviting people to come to the meetings for healing.

Good morning doc, I remember you mentioning this outfit in one of your blogs but I would like to know more about dr. Neil Riodan and the macenna institute. I attended one of their marketing meetings in ohio and it was the wild west of stem cells. The speaker claimed that riodan had 40 patents and 70 publishings to back up all of his claims. I wont bore you with all the claims, you have heard them before, but he made one remark about no possibility of rejection using foreign live cells because of the HLA protein. I did not know what he was talking about and tried to research it and did not find anything to explain it. I will add that the procedures range from $4000 to $14000 administered in your home by a PA. At the end of the meeting I confronted the speaker about all the stuff I learned from you and he said Regenexx is a bully. Is there any truth about the HLA protein?

Thats a good question. On the website, they claim to offer both Regenerative Cell Therapy and be a Leader in Joint Pain Therapy. Their website is pretty nebulous, but lets start with this coverage map:

That word choice is interesting. Notice how instead of saying that the company has affiliates, offices, or clinics in these states, it uses the term coverage. In fact, you cant find out anything about what this coverage entails. However, a familiar name does pop up here on their website: Scott Gray, DC.

Ive blogged on Scott Gray, DC before. He is/was the CEO of Stem Cell One and Gray Marketing Enterprises. What is Stem Cell One? Another chiropractic clinic that has billed itself as Leading Umbilical Stem Cell Therapy in Marion, Ohio! Scott also owns a company called Gray Marketing Enterprises, and he used to state on his LinkedIn page, Dr Gray also published a book helping chiropractors help more patients; The Ultimate New Patient Machine. That profile, after being discussed in a prior blog has now been deleted.

Ive also blogged before on Biologics Health, a company associated with Scott Gray. When you Google Biologics Health now and Scotts name, Riverside Biologics Health comes up, so I have to assume that Scott added the word Riverside to this company.

Last year when I reviewed Scotts at-home umbilical cord stem cell service called Biologics Health, it was pretty much the same business now being described by this patient. A nurse would come to your home to give you an IV of umbilical cord stem cells. It also looks like that same nurse will perform a joint injection, but perhaps thats performed in a clinic? This was all very concerning at that time, as any such procedure should be performed in a room that looks like this:

And not like this:

Another problem was that the research back then and since, given our new paper published in the American Journal of Sports Medicine and those papers published by others, showed that commercially available umbilical cord preparations had no living stem cells (1-4). It looks like since the FDA crackdown Scott has scrubbed this new website of the phrase stem cells, but based on the patients communication above, thats still central terminology used in their seminars. Frankly, its amazing that the FDA has yet to stop this one, as IMHO I can think of few things that sound more innocuous, but in my opinion, are more dangerous than an IV umbilical cord infusion. Lets dig into that topic.

First, as discussed above, none of these umbilical cord products sold in the US have any live and functional mesenchymal stem cells. The research in this area has made that clear time and time again. However, a few of these products do have live and dying cells which are foreign tissue. When umbilical cord blood treatments are used in pediatric cancer cases, these are HLA matched to the patient (5-7). Why? The patients immune system will reject the cells, however, there will be less rejection if the cell surface markers on the umbilical cord cells are a close match to the patients own markers. However, I have yet to see a company using this stuff that matches the patient to the product.

What can happen when mismatched umbilical cord cells are injected into a body? A severe rejection reaction known as Graft vs. Host Disease (GVHD) (7). GVHD can be mild with rashes and feeling sick or it can be severe with organ failure. I know this one personally, as Im the medical expert on several legal cases where umbilical cord tissue injections were given in chiropractic and medical offices, and in my medical opinion, caused GVHD. In one case, a poor elderly man went into renal failure.

How about the salespersons statement above? This what the patient relayed was said:

One item of interest I did not understand was that the DNA becomes inactive when the cells are extracted from the fetus because of the HLA protein so there is no possibility of rejection.

Is this scientifically accurate? Nope. If you have live cells that are dying, the tissue will be read as a foreign invader by the patient and an immune response will be mounted. Now if the manufacturer made a conscious effort to kill all of the cells, that could help. However, the handout given out at the seminar by Riverside Biologics Health website says:

That certainly sounds like Riverside is claiming live cells. The rest of the brochure talks about how stem cell numbers decline with age (not actually accurate in the way portrayed, see this blog for more information).

Who gave this medical advice? Scott Boyer, a guy with a 4-year communications degree and Dale Carnegie sales training. Based on his Linkedin profile, Scott is a professional salesperson experienced in giving sales presentations and NOT a medical or scientific expert.

One of the bait and switch tactics used by these stem cell outfits is that they post research that has nothing to do with the product theyre offering to lend a veneer of credibility to their sales pitch. Riverside Biologics Health does that as well. Not a single paper listed has anything to do with the off-the-shelf umbilical cord tissue product theyre using. Even nuttier, they have listed one of my earliest research papers on using culture-expanded bone marrow stem cells in a knee, which again has nothing to do with dying umbilical cord tissue in a bottle.

Its clear from the patient that Neil Riordans product is being used in this venture. This is a product that I have blogged on called Signature Cord where the manufacture has claimed that it contains live and functional mesenchymal stem cells. Again, despite this past claim, there is no credible scientific data that this umbilical cord product contains any live and functional MSCs.

I know Neil. He has a Ph.D. and is a Physicians Assistant. He has published a slew of things about his live stem cell product being used at his clinic in Panama but has yet to publish any randomized controlled trial on those treatments. In addition, I know of no studies at all on the US product called Signature Cord, which is whats being used here. Hence, in my opinion, this is more bait and switch on the part of Riverside.

Given that Scotts Linkedin profile was deleted, I decided to see if I could find a corporate HQ for either Biologics Health or Riverside Biologics Health. That led me to a corporate registration agent in St. Pete, Florida, but no offices could be found. IMHO, thats par for the course here, as Riverside is a sales company run by a chiropractor whose career has focused on increasing the revenue of chiro practices. Meaning that Riverside is not a biotechnology venture nor is it a chain of clinics staffed by physician specialists.

The website states, the products of conception (amniotic fluid, umbilical cord, placenta, and accompanying materials) are discarded. In this case, its donated by the mother, with no harm to the baby. The materials are transferred immediately via a sterile container to a nearby FDA certified lab.Is there such a thing? Nope. The FDA doesnt certify labs that process birth tissues. You can register your lab via a free registration system (Tissue Establishment Registration), but that only tells them where to go if they want to inspect you. That doesnt mean they have certified or approved your lab.

One of the reasons that there has been a name change, adding the word Riverside to Biologics Health may be that the company received a letter from the FDA. This letter states exactly what I have reviewed, that Riverside Biologics Health is violating federal law by delivering umbilical cord IV treatments to patients. Has that stopped the company? Apparently not as they have given seminars after that date.

The upshot? At the end of the day, IMHO, sending nurses to homes to inject umbilical cord tissue is not Kosher. I hope the FDA continues to act on this one.

______________________________________

References:

(1) Berger D, Lyons N, Steinmetz, N. In Vitro Evaluation of Injectable, Placental Tissue-Derived Products for Interventional Orthopedics. Interventional Orthopedics Foundation Annual Meeting. Denver, 2015. https://interventionalorthopedics.org/wp-content/uploads/2017/08/AmnioProducts-Poster.pdf

(2) Becktell L, Matuska A, Hon S, Delco M, Cole B, Fortier L. Proteomic analysis and cell viability of nine amnion-derived biologics. Orthopedic Research Society Annual Meeting, New Orleans, 2018. https://app.box.com/s/vcx7uw17gupg9ki06i57lno1tbjmzwaf

(3) Panero, A, Hirahara, A., Andersen, W, Rothenberg J, Fierro, F. Are Amniotic Fluid Products Stem Cell Therapies? A Study of Amniotic Fluid Preparations for Mesenchymal Stem Cells With Bone Marrow Comparison. The American Journal of Sports Medicine, 2019 47(5), 12301235. https://doi.org/10.1177/0363546519829034

(4) Berger DR, Centeno CJ, Kisiday JD, McIlwraith CW, Steinmetz NJ. Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics. Am J Sports Med. 2021 Oct;49(12):3404-3413. doi: 10.1177/03635465211031275. Epub 2021 Aug 16. PMID: 34398643.

(5) Eapen, Mary et al. Mismatched Related and Unrelated Donors for Allogeneic Hematopoietic Cell Transplantation for Adults with Hematologic Malignancies. Biology of Blood and Marrow Transplantation, Volume 20, Issue 10, 1485 1492. https://www.ncbi.nlm.nih.gov/pubmed/24862638

(6) Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104(7):19231930. https://www.ncbi.nlm.nih.gov/pubmed/15191952

(7) Holtan SG, Pasquini M, Weisdorf DJ. Acute graft-versus-host disease: a bench-to-bedside update. Blood Jul 2014, 124 (3) 363-373; DOI: 10.1182/blood-2014-01-514786

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Riverside Biologics Health Review: At Home "Stem Cells ...