Differentiating Hope from Embryonic Stem Cells

Courtesy of Nadya Lumelsky and Ron McKay PANCREATIC CELL PUZZLE: A cell sub-population in differentiated embryonic stem cell cultures produces pancreatic islet hormones, insulin (red) and glucagon (green). Although most cells produce only one type of hormone, the cells shown in yellow produce both. The co-production of the two hormones might signify developmental immaturity. The ethical dimension of embryonic stem cell research looms so large in the public consciousness that the underlying science often gets taken for granted. But within the ESC research community, realism has overtaken early euphoria as scientists realize the difficulty of harnessing ESCs safely and effectively for clinical applications. After earlier papers in 2000 and 2001 identified some possibilities, research continued to highlight the tasks that lie ahead in steering cell differentiation and avoiding side effects, such as immune rejection and tumorigenesis. In 2001, several seminal papers laid much of the groundwork, and this issue's Hot Papers were particularly influential. Nadya Lumelsky at the National Institutes of Health and colleagues described the generation of cells expressing insulin from murine ESCs.1 Lumelsky's group, unlike the researchers of the other two Hot Papers, was precluded from using human ESCs under NIH funding rules. The second paper by Su-Chun Zhang and researchers at the University of Wisconsin and the University of Bonn, Germany, describes the in vitro formation of neural tube-like structures from differentiating human ESCs.2 The final paper, based on research at the Technion-Israel Institute of Technology in Haifa, showed how human ESCs could differentiate into cardiomyocytes.3 With the correct stimuli, these cells could pulsate in the manner expected of early-stage cardiac muscle. Despite continuing debate over the validity of some of the experimental protocols, the papers have been widely acknowledged for identifying clinical possibilities. "Their findings are particularly encouraging regarding the prospect of applying embryonic stem cells in a clinical context for repair of damaged heart muscle, restoring insulin production in diabetics, and for combating neurodegenerative diseases," says Richard Gardner, a stem cell specialist at the University of Oxford. Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. N. Lumelsky et al., "Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets," Science, 292:1389-94, 2001. (Cited in 161 papers) S.C. Zhang et al., "In vitro differentiation of transplantable neural precursors from human embryonic stem cells," Nat Biotechnol, 19:1129-33, 2001. (Cited in 84 papers) I. Kehat et al., "Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes," J Clin Invest, 108:407-14, 2001. (Cited in 99 papers) PICKING ON PROTOCOLS Further research, both by the teams involved and by others attempting to replicate the results, improved the protocol, or in a few cases identified shortcomings in methods used. Lumelsky's research generated the greatest controversy when, earlier this year, Douglas Melton and postdoc Jayaraj Rajagopal at Harvard University cast doubt on the results.4 Melton's group, working with Lumelsky to repeat the protocol, could not detect mRNA from insulin1 or 2 in the cultures. The cells apparently failed to transcribe a lacZ insertion downstream of endogenous insulin genes, and other evidence for endogenous insulin production was missing. They suggested that Lumelsky's cells were merely masquerading as pancreatic cells by absorbing insulin from the culture rather than producing it. It was a possibility that Lumelsky says she hadn't considered. The foundation for the Hot Paper was that protocols producing neural cells from murine ESCs could be adapted to yield endocrine pancreatic cells. She suggested in the paper that nestin, an intermediate filament protein normally occurring in neural precursors, might be an important step in that enrichment process, but whether nestin is a marker of the pancreatic progenitors is not proven. "There is a lot of variability in those cultures," Lumelsky adds, an uncertainty that Melton's group concedes in their paper. Lumelsky agrees that, in general, critics have successfully highlighted the problems to be surmounted before attempting clinical application of ESCs. "But we stand by our work," Lumelsky says, citing papers that have since repeated her results.5,6 Such work appears to support the study's conclusions, but not the methods. "I would say our results confirmed the data, but not the protocol," says researcher Przemyslaw Blyszczuk of Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany, first author on one paper5 repeating Lumelsky's work. Blyszczuk and others confirmed that insulin-producing pancreatic cells had been derived, but disagreed that nestin-positive cells were the predominant precursors. "You can get some pancreatic differentiation from the nestin-positive cells, but these are not the main progenitor cells," says Blyszczuk. Lumelsky says, "Culturing under conditions which promote generation of nestin-expressing cells allowed us to make more insulin producing cells." But, she adds, "We don't claim that nestin-positive cells are progenitors." Blyszczuk and others have been working to identify the main progenitor cells. He says a paper slated for publication in the spring will reveal the outcome of this research. The controversy highlights the greater challenge of identifying clearly the events leading to fully differentiated cells in vitro. Although the other two papers attracted less controversy, their authors also had to show that their results were really the product of differentiated ESCs. TUBULAR RESULTS The Zhang paper on neural cells2 provided the first example of differentiating human ESCs, says coauthor Ian Duncan. But Zhang, an assistant professor at Wisconsin, was most excited by the tube-like rosette structures similar to those produced through neural differentiation. "The cells organized themselves into neural tubes, and always did so," says Zhang. Courtesy of Su-chun Zhang ROSETTES ARE FREE: Neural tube-like rosettes developed from human embryonic stem cells. The bottom panel shows magnified view of columnar cells that comprise the rosettes. The team then repeated the experiment in vivo with mice and observed similar neural-tube structures being formed, even though this had not been reported from previous experiments with murine ESCs. "The interesting thing is that mouse embryonic stem cells have been studied for two decades," says Zhang; he ascribes the inability to produce such tube structures to the protocols used. "[Researchers] drove the cells to a very differentiated state, and so you end up with a very mixed population.... Then it becomes difficult for the cells to be useful either as a tool for development study or as a source for stem cell therapy." The therapeutic potential of Zhang's work lies in curing human neurodegenerative diseases such as Parkinson. Lars Björklund at Harvard Medical School and others have reported some success in treating these diseases in animal models.7 But Zhang notes that there is a long way to go in improving the protocol so that the cellular and molecular mechanisms can be analyzed more rigorously, and also in providing a reference model for further research. HEART-MUSCLE MENDING Different concerns confronted Izhak Kehat and his team in the wake of their Hot Paper, which demonstrated the differentiation of human ESCs into cardiac myocytes. "It was the first time that human heart cells that were not taken out of the human heart were grown in a laboratory," says Kehat. The therapeutic potential here lies in the possibility of transplanting cells to repair damaged or dead heart tissue, alleviating the need for whole-organ donors. This already has been attempted with ESCs in animal models and with adult stem cells in patients with advanced disease. Kehat says that ESCs offer the greatest promise for cardiac therapies, as trials with adult stem cells have yet to deliver consistent results. Kehat concedes that several challenges face the use of ESCs, notably immune rejection. He mentions two strategies to combat this: "One is to make a lot of stem cells and create a bank with 200 or 300 immunological types," which would cover most of the immunological angles but would require careful screening. The other, more controversial possibility would use therapeutic cloning to create an embryo from the patient's own cells, which would provide a source for ESCs. "If this is possible and ethically acceptable, it could make a lot of replacement parts," says Kehat. But the challenge of tumor formation must be overcome before embryonic cells can be used for therapy. Protocols to develop highly differentiated offspring cells for transplant may combat this, says Duncan. Perhaps the greatest challenge lies in establishing, beyond all reasonable doubt, that therapeutic benefits observed in experimental animals really do result from stem-cell differentiation. As Lumelsky points out, the need exists to identify individual cells through surface markers and to track them through the entire differentiation process, a process possibly wrought with as much controversy as ESCs themselves. Philip Hunter (ph@philiphunter.com) is a freelance writer in London. References 1. N. Lumelsky et al., "Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets," Science, 292:1389-94, 2001. (Cited in 161 papers) 2. S.C. Zhang et al., "In vitro differentiation of transplantable neural precursors from human embryonic stem cells," Nat Biotechnol, 19:1129-33, 2001. (Cited in 84 papers) 3. I. Kehat et al., "Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes," J Clin Invest, 108:407-14, 2001. (Cited in 99 papers) 4. J. Rajagopal et al., "Insulin staining of ES cell progeny from insulin uptake," Science, 299:363, 2003. 5. P. Blyszczuk et al., "Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells," Proc Natl Acad Sci, 100:998-1003, 2003. 6. Y. Hori et al., "Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells," Proc Natl Acad Sci, 99:16105-10, 2002. 7. L.M. Björklund et al., "Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model," Proc Natl Acad Sci, 99:2344-9, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Differentiating Hope from Embryonic Stem Cells

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Courtesy of Nadya Lumelsky and Ron McKay
PANCREATIC CELL PUZZLE: A cell sub-population in differentiated embryonic stem cell cultures produces pancreatic islet hormones, insulin (red) and glucagon (green). Although most cells produce only one type of hormone, the cells shown in yellow produce both. The co-production of the two hormones might signify developmental immaturity.