26th Annual International Symposium Stem Cells: Biology and Applications Overview The potential of stems cells and induced pluripotent stem cells iPSCs to be differentiated and returned to one’s own body as a therapeutic source of “replacement” cells or tissue is on the horizon. The 26th Annual International Symposium of the Center for Study of Gene Structure and Function described the basic science of iPSC and other stem cell developments, pioneering experiments and ethical implications. In one morning and two afternoon sessions, ten scientists discussed their basic and/or clinical research, and one distinguished medical ethicist/writer discussed participation, benefits, and risks of stem cell research. The morning session focused on the ability to derive pluripotent stem cells and their ability to be differentiated into specific populations for therapeutic purposes. The first afternoon session delved into the applied science aspects of directed differentiation and the ethical implications of obtaining biological material and reintroducing modified versions back into patients. The second afternoon session started by discussing the mechanism of tissue regeneration in an invertebrate with its potential implications for cell replacement in mammals by reinvigorating inherent stem cell populations. The symposium concluded by discussing tissue engineering or synthesis in the lab with the possibility of reintroduction into the body. Sponsorship This conference and meeting report were made possible with support from the Weill Cornell Medical College, Clinical & Translational Science Center (CTSC), Hunter College of the City University of New York, the National Institutes of Health, National Institute on Minority Health and Health Disparities, Research Centers in Minority Institutions – 8 G12 MD007599-27 and the Clinical and Translational Science Awards – 2UL1TR000457-06.

Introduction

The Generation of Human Induced Pluripotent Stem Cells The stem cell symposium began with a presentation from the person who derived the first human embryonic stem cell line in 1988: James Thomson, from the Morgridge Institute for Research at the University of Wisconsin. His lab was also among the first to generate human induced pluripotent stem cells, in 2007. Thomson spoke of the genetic and epigenetic differences between these two populations of pluripotent cells, some of which came as a surprise to researchers and all of which must be taken into account before any of the cells are put to clinical use. He concluded by highlighting areas in which the cells might be best utilized, notably in studying embryonic development and neurodegenerative diseases and as a model system to screen cardiac drugs for safety and efficacy. “If I Only Had A Heart” Thomson‟s talk segued beautifully into that of Christine Mummery, from Leiden University Medical Center in the Netherlands, who expanded on the notion of differentiating human induced pluripotent stem cells into cardiomyocytes as a model system for drug screening. She and others have shown that iPSC- derived cardiomyocytes beat in tissue culture plates, and thus EKG-like waves comparable to real EKGs can be taken on them under different conditions – for instance, in the presence of different doses and combinations of drugs – to determine how these conditions affect heart function. This methodology can be especially useful for individuals with rare genetic mutations, and might be one of the first steps to truly personalized medicine. Stem Cell Therapy Lorenz Studer, who works just three blocks away from Hunter at Memorial Sloan Kettering, was the first speaker of the day to mention putting stem cells into patients. He works with people who have Parkinson‟s disease, which is an appealing target for stem cell therapy because it cannot be cured and because it is caused by the loss of a single cell type: dopamine neurons. These cells originate during embryonic development and are not regenerated over an individual‟s lifetime; the only way to deal with their loss is to introduce new ones. Studer has successfully cured mice with Parkinson‟s and is currently testing a number of embryonic stem cell lines for use in humans. Cell Fusion Hunter‟s own Paul Feinstein wrapped up the day‟s first session. His main interest is in olfaction, and studying olfaction requires a lot of transgenic mice. In making all of these mice, Feinstein realized that making embryonic stem cells is very labor and time intensive, and that the process generates many aneuploidies. He thought there might be a quicker way to assay for these errors. His lab recently developed a fast and accurate screen for detecting common aneuploidies in ESC and iPSC using quantitative real time PCR. He is also exploring easier ways of developing human induced embryonic stem cells via cell fusion.

Whoa, Not So Fast Harriet Washington, the author of Medical Apartheid: The Dark History of Medical Experimentation on Black Americans from Colonial Times to the Present, opened the afternoon session on the broader social implications of stem cell research with a large dose of warning and perspective. She noted that the medical establishment has historically been less than forthcoming with patients, often – but not only – when those patients were poor and downtrodden. She stressed that patient consent is absolutely essential in all medical testing and technological developments, even if that means we must forego potentially valuable advances because attaining proper consent is not feasible.

Bone Marrow Transplants Although Thomson started the day by opining that stem cell therapy is still many years in the future, Koen van Besien, of Weill Cornell Medical College, reminded us of one area in which it has a tried and true history: treating hematopoietic disorders, especially leukemia. In 1968 the Human Leukocyte Antigen (HLA) system was identified as the major determinant of graft rejection or success. van Besien noted that hematopoietic stem cells in umbilical cord blood are more immunologically naïve than those in adult bone marrow, and they are more tolerant. Thus, when transplanted, they do not need to match the recipient as well as adult cells do in order to avoid graft versus host disease. Treating HIV The next speaker was Paula Cannon from USC‟s Keck School of Medicine. The proof of principle underlying her work comes from the Berlin Patient, a man with AIDS who developed leukemia and was cured of both conditions with transfusion of CCR5-/- T cells, which are resistant to HIV. He is the only adult to date who has been cured of AIDS. Cannon uses zinc finger nucleases to disrupt the CCR5 gene in hematopoetic stem cells taken from AIDS patients to render the cells, and hopefully the people they came from, impervious to HIV. She chose to engineer hematopoietic stem cells, rather than mature T cells, because then all of their progeny would receive protection: T cells, macrophages, dendritic cells, and microglia. All of these cells can be targeted by HIV.

Locus Control Regions Benjamin Ortiz, from Hunter, ended the first afternoon session. He studies the T cell receptorLocus Control Region (LCR). LCRs are different from other cis-acting transcriptional regulatory regions, like promoters and enhancers, in that they can confer both copy number dependence and lineage specific expression to a linked transgene in mice. These features make them extremely valuable for the prospect of gene therapy. Like the olfactory system studied by Paul Feinstein, the best model system to study the TCR LCR has thus far been transgenic mice. Past literature had suggested that establishing an LCR‟s function may require it to be present in the genome before differentiation. Ortiz hypothesized that introducing the LCR into mouse embryonic stem cells, that are then directed to differentiate into T cells in vitro, might enable recapitulation all of its characteristics. The in vitro system he developed based on this insight supports all of the features of the LCR that make it so useful.

Regeneration and Planarians The day‟s final session focused on the use of stem cells in engineering, and was opened by Alejandro Sánchez Alvarado from the Stowers Institute for Medical Research. Alvarado is interested in the evolutionary origin of stem cells, which are present in most multicellular organisms. He studies embryonic development in the freshwater planarian (flatworm) Schmidtea mediterranea. Planaria can regenerate after amputation, and this regenerative capacity is due to the presence of self-renewing, undifferentiated cells called neoblasts. Data from Alvarado‟s lab suggest that neoblasts arise during a certain defined point during development and represent a specialized cell fate. More Cardiac Drug Screening Like Thomson and Mummery in the morning session, Peter Zandstra from the University of Toronto spoke of using cardiomyocytes derived from stem cells to screen cardiac drugs for safety and efficacy. But whereas they had petri dishes of beating cardiac cells, Zandstra‟s lab has made arrays of cardiac micro tissues embedded in three-dimensional micro patterned matrices. By varying the conditions under which he makes these micro tissues, Zandstra hopes to find the conditions that will turn stem cells into myocardial tissue that will be optimal for transplantation. He is also using computational analysis of growth conditions by which many complex factors control hematopoiesis. This process should scale up the production of hematopoetic stem cells from cord blood, because although these cells are desirable for a number of clinical applications, they are seldom available in sufficient amounts. Hearts and Bones Gordana Vunjak-Novakovic, from Columbia University, concluded the day‟s talks. She discussed how tissue engineers can use stem cells to create the “spare parts” we may need as our life spans extend, and spoke of her work in developing scaffolds and bioreactors, which are as vital as the cells themselves in bioengineering cell culture environments to make these spare parts. She has been concentrating on making a properly sized, anatomically shaped, viable temporomandibular joint (TMJ) using human mesenchymal stem cells to treat injuries to the head and face. Experiments are currently under way to test these bone grafts in pigs.

MORNING SESSION: Pluripotency and Directed Differentiation Human Pluripotent Stem Cells

Speaker: James Thomson, Director of Regenerative Biology, Morgridge Institute for Research, University of Wisconsin Highlights

• Human ES/iPS cells provide a tissue culture model for understanding the differentiation and function of human tissues.

• Human ES/iPS cells could provide purified populations of specific differentiated cells for drug discovery and testing.

• Human ES/iPS cells could provide a potentially unlimited source of cells for transplantation therapies.

• However, human ES and iPS are not identical to each other, or to their murine counterparts.

ES vs. iPS cells Mice are great lab animals, but there are limits to what they can model. For example, mouse and human development are quite different, especially at the early post implantation stage. Thus, researchers have been searching for a better model of early human development. To this end, embryonic stem (ES) cells were isolated from rhesus monkeys and marmosets in 1995, and then Dolly the sheep was cloned in 1997 – demonstrating that the differentiated state of somatic cells is reversible.

Human ES cells were isolated in 1998. During the first week of development, while these cells are dividing by cleavage, they do not differentiate. Cells do not become committed to their fates until after they implant into the wall of the uterus. So these ES cells are pluripotent- they can form any cell of the body - and they self-renew in culture forever (or at least for 500 doublings, which is the most anyone has bothered to record. Skin cells senesce after 70 doublings.) Unlike cancer cells though, which also self-renew in culture forever, ES cells are normal. They replicate without limit because unlike somatic cells they still have active telomerase.

In 2007, Dr. Thomson’s lab reported on their generation of human induced pluripotent stem cells (iPSC) [5]. These cells have normal karyotypes; they have telomerase activity; they express cell surface markers and genes that characterize human ES cells; and they maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers (endoderm, mesoderm, and ectoderm).

Yet important differences remain between human iPSC and human ES, at both the genetic and epigenetic levels. Genetic mutations creep in by a selection process that is a byproduct of reprogramming, regardless of the reprogramming method used [2]. Thomson also found iPS-specific methylation that was seen in neither ES cells nor the progenitor cells from which the iPSC were made [3]. Some of these differentially methylated regions reflect a memory of the somatic tissue of origin, and others may indicate that certain genomic loci, notably those near the telomeres and centromeres, might have a tendency to undergo inappropriate reprogramming of CG methylation. Thomson highlighted that before any sort of clinical use, stem cells must be screened for genetic mutations to make sure they are safe and to minimize any risk that they might cause cancer.

As noted above, early post implantation human development is inaccessible to study and different from the mouse. Yet studying development is not the only aspect of basic research that can utilize ES and iPSC. Thomson spoke of human iPSC that have been differentiated in vitro into motor neurons that undergo degeneration, and thus can be used as a model system to study neurodegenerative diseases and macular degeneration. In addition, he talked about human iPSC that have been differentiated into cardiomyocytes, and provide a more accurate model for drug screening than any other system available. These cells can be used not only to screen for drugs that might be effective, but also to make sure that they are safe. These cells beat in their dishes, and were more predictive of drugs that would cause cardiac arrhythmia – a common reason that approved drugs must be pulled from the market – than other models currently in use, such as screening the drugs on live dogs.

Transplantation therapies based on ES/iPS cells bear “eerie parallels to gene therapy,” said Thomson. “Ultimately they will change the world. But they will take time. And they will initially fail.” References [1] Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, Smuga-Otto K, Howden SE, Diol NR, Propson NE, Wagner R, Lee GO, Antosiewicz-Bourget J, Teng JM, Thomson JA. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011 May; 8(5):424-9. doi: 10.1038/nmeth.1593. Epub 2011 Apr 10.

[2] Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, Zhang K. Somatic coding mutations in human induced pluripotent stem cells. Nature. 2011 Mar 3; 471(7336):63-7. doi: 10.1038/nature09805. [3] Lister R, Pelizzola M, Kida YS, Hawkins RD, Nery JR, Hon G, Antosiewicz-Bourget J, O'Malley R, Castanon R, Klugman S, Downes M, Yu R, Stewart R, Ren B, Thomson JA, Evans RM, Ecker JR. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 2011 Mar 3; 471(7336):68-73. doi: 10.1038/nature09798. Epub 2011 Feb 2. [4] Yu J, Vodyanik MA, He P, Slukvin II, Thomson JA. Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion. Stem Cells. 2006 Jan; 24(1):168-76. Epub 2005 Oct 6. [5] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec 21; 318(5858):1917-20. Epub 2007 Nov 20.

Directed Cardiac Differentiation of Pluripotent Stem Cells and Potential Applications

Speaker: Christine Mummery, Professor of Developmental Biology and Chair of the Department of Anatomy and Embryolog, Leiden University Medical Center

Highlights

Induced pluripotent stem cells can be “the new patient,” as diseases can be studied and treated in tissue culture dishes.

Technological improvements are allowing the generation, differentiation, and scaling up of induced pluripotent stem cells, and are also enabling the establishment of appropriate controls.

Patient- derived human induced pluripotent stem cells will hopefully one day usher in a new type of personalized medicine.

Regenerating Cardiomyocytes

Induced pluripotent stem cells (iPSC) are generated by isolating somatic cells and reprogramming them through the ectopic expression of four transcription factors. After reprogramming, the iPSC can be grown in different types of media to induce differentiation into specialized cell types that mimic mature cells. Because the iPSC retain the genetic profile of the somatic cell from which they were generated, they can be used to study rare genetic diseases. They are also valuable for studying diseased tissue that is difficult to obtain via biopsy, such as that in the heart and the brain.

Advances in inserting genes into human iPSC and embryonic stem cells (ESC) have been invaluable in establishing controls for such studies. Thus, if studying a disease using iPSC from a patient with a genetic disease, it is now possible to correct the genetic defect in these cells for use as controls. There are even banks of iPSC with genetic defects being developed – along with their cognate genetically repaired control cells [1].

When differentiating iPSC into cardiomyocytes, researchers take their lessons from embryonic cardiac development [3]. The cells must be exposed to three families of protein growth factors to mimic the early stages of mesoderm formation and cardiogenesis - bone morphogenic proteins, WNTs, and fibroblast growth factors – and they must be exposed to them in similar spatial and temporal gradients as they would in a developing embryo. So when the growth factors are removed is as important as when they are administered. These growth factors induce the expression of various transcription factors in a sequential manner to generate different types of cardiac cells. Ideally, Dr. Mummery said, differentiating cardiomyocytes from iPSC will one day be automated, robust, inexpensive, and reproducible: “like a cake mix: just add water.”

As the iPSC are differentiating, it is not necessarily so trivial to determine if they are differentiating along the desired path. Cardiac cell types are best distinguished by the presence of transcription factors or cytoplasmic proteins. Mummery found that VCAM-1 is an accurate cell surface marker that can be used to track differentiation, as it is present only in embryonic cardiomyocytes and not their progenitors. Once the different types of heart cells – cardiomyocytes, endothelial cells, and smooth muscle - have all differentiated properly, they beat in the dish just as they would in the body.

Drug screening, discovery, interaction, and toxicity

Mummery put her iPSC-derived cardiomyocytes into mice in which myocardial infarctions had been induced – and although they started to graft in nicely, they didn’t ultimately help the mice. Possibly because they were human cells; possibly because human hearts beat 60 times per minute and mouse hearts beat 500 times per minute. Regardless, this experiment demonstrates that mice are not an ideal model for studying and treating the cardiovascular disease that plagues much of the Western world.

The QT interval on an electrocardiogram (EKG) is one way of telling if the heart is behaving normally. It can be modified by genetic defects or by drugs, and although a long QT interval can lead to ventrical tachycardia and death, it is not necessarily associated with concomitant cardiac structural abnormalities. Many drugs are removed from the market because they cause arrhythmias, as indicated by a change in the length of the QT interval. Because iPSC- derived cardiomyocytes beat in tissue culture plates, EKGs can be taken on them under different conditions – for instance, in the presence of different doses and combinations of drugs – to determine how they affect heart function. In this way, compounds can be screened for both efficacy and safety, and this system better recapitulates the situation in vivo than any other method currently in use. This methodology can be especially useful for individuals with rare genetic mutations the ramifications of which might not be known, or for people who must simultaneously take multiple medications. Already, it has shown that some of these drugs cause arrhythmias specifically in patients deficient in a particular potassium channel. “The use of human iPS cells could contribute to a more efficient development of new drug candidates, rescue those that may have unnecessarily been withdrawn because of negative effects in non-optimal assays and meet the significant need for protective predictive toxicology bioassay systems that more closely approximate effects observed in humans, particularly identifying susceptible groups,” wrote Mummery [1]. Her work on iPSC- derived cardiomyocytes is bringing all of those goals closer to reality.

References

[1] Bellin M, Marchetto MC, Gage FH, Mummery CL. Induced pluripotent stem cells: the new patient? Nat Rev Mol Cell Biol. 2012 Nov; 13(11):713-26. doi: 10.1038/nrm3448. Epub 2012 Oct 4.

[2] Davis RP, Casini S, van den Berg CW, Hoekstra M, Remme CA, Dambrot C, Salvatori D, Oostwaard DW, Wilde AA, Bezzina CR, Verkerk AO, Freund C, Mummery CL. Cardiomyocytes derived from pluripotent stem cells recapitulate electrophysiological characteristics of an overlap syndrome of cardiac sodium channel disease. Circulation. 2012 Jun 26; 125(25):3079-91. doi: 10.1161/CIRCULATIONAHA.111.066092. Epub 2012 May 30. [3] Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res. 2012 Jul 20; 111(3):344-58. doi: 10.1161/CIRCRESAHA.110.227512. [4] Mummery CL, Passier R. New perspectives on regeneration of the heart. Circ Res. 2011 Sep 30; 109(8):828-9. doi: 10.1161/RES.0b013e3182349a8a. [5] Mummery CL, Lee RT. Is heart regeneration on the right track? Nat Med. 2013 Apr; 19(4):412-3. doi: 10.1038/nm.3158.

Human Embryonic Stem Cell Derived Midbrain Dopamine Neurons for Cell Therapy in Parkinson’s Disease

Speaker: Lorenz Studer, Director, Center for Stem Cell Biology, Memorial Sloan Kettering Cancer Center

Highlights

Parkinson’s disease is an attractive target for stem cell therapy because it is caused by the loss of a specific cell type: dopamine neurons.

The donor cells must come from the floor plate, a specific part of the midbrain. Grafting intermediate stage cells offers the best compromise of cell survival and

integration.

Parkinson’s disease

Parkinson's disease is a progressive disorder of the nervous system that affects movement, causing tremors, stiffness, and slowness that become more and more debilitating with time. Although medical and surgical treatments can mitigate the symptoms, Parkinson’s disease cannot be cured. Annual health care expenses for surgical therapies of Parkinson’s disease run to six million dollars in North America alone. The key motor symptoms that characterize Parkinson’s disease are caused by the loss of midbrain dopamine neurons. Healthy people have about half a million of these cells; about half of them are already gone before symptoms become apparent. “This is a really big problem,” said Dr. Studer, because these cells are born during embryonic development, approximately six to eight weeks post fertilization, and no more are made after that. Parkinson’s disease was thus one of the first ailments to be considered for a good candidate for treatment with stem cell therapy; the only way to get functional dopamine neurons back is to reintroduce them. Human fetal tissue was initially considered as a source for these cells, and about in the 1980’s about 300 patients received fetal tissue grafts. When they were reintroduced into adult brains, they could still make dopamine; but they did not save all of the patients. Stem cells were thus considered as an alternate source. Which cells should we put in, and at what point in their development?

Dopamine neurons were generated from human induced pluripotent stem cells; they looked great in the dish, expressing all of the proper markers, but unlike human fetal cells, none survived when they were transplanted into mouse models of Parkinson’s disease. In 2010, Studer’s lab had the breakthrough realization that the stem cells needed to come from a very particular region of the midbrain in order to completely recapitulate the phenotype they needed; they needed to come from the floor plate, a signaling center during human neuron development that is located along the ventral midline of the developing embryo [1]. Once they realized this, they could make a constant supply of dopamine neurons that could function as a model system to study the early stages of human neural development, which had heretofore been inaccessible. These cells survived for months in mice and monkeys, and rescued some of the animals’ behavioral defects. These cells have now been generated in a number of different laboratories, confirming the validity of the results.

But before using the cells clinically in humans, they wanted to determine at which stage of their development the cells should be introduced. Transplanting any undifferentiated,

pluripotent cells into humans always comes with the risk that they will turn into tumors. Studer’s group examined mouse ESC derived dopamine neurons at three different stages of differentiation – early, middle, and late - to identify which would be optimal for engraftment. They found that cells at the intermediate stage of neuronal differentiation offered the best compromise of getting good survival but integration into the mouse brain and good functional recovery [2]. Interestingly, the engrafted cells not only replenished the missing dopamine neurons in the Parkinsonian mice, they also culled the excess serotonin and GABA neurons that had grown in to fill the void left by the degenerated dopamine neurons. The clinical implications of this finding are not yet fully understood.

Of course, Studer pointed out, curing humans will not be the same as curing mice. One issue is that the human brain is larger, and the dopamine neurons will therefore have to grow accordingly longer axons. They must therefore be treated with a “molecular lubricant” – an enzyme called PSA important in neural development that increases cell adhesion. Because these are postmitotic neurons that no longer proliferate, the risk of tumor formation should be minimal; but Studer spoke of genetically engineered safety switches that would eliminate any proliferating cells anyway. Such switches might also be used to prune the grafts should that become necessary.

Human embryonic stem cells, in contrast to induced pluripotent stem cells, have already been approved by the FDA for use in humans to treat macular degeneration. Thus Studer is currently testing a number of ESC for use in treating Parkinson’s, and he is also examining cells known to be suited to make dopamine neurons that have not yet been used in humans. He hopes to be in clinical trials by 2016.

References

[1] Fasano CA, Chambers SM, Lee G, Tomishima MJ, Studer L. Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell. 2010 Apr 2; 6(4):336-47. doi: 10.1016/j.stem.2010.03.001. [2] Ganat YM, Calder EL, Kriks S, Nelander J, Tu EY, Jia F, Battista D, Harrison N, Parmar M, Tomishima MJ, Rutishauser U, Studer L. Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment. J Clin Invest. 2012 Aug 1; 122(8):2928-39. doi: 10.1172/JCI58767. Epub 2012 Jul 2.

Generation of Induced Pluripotent Stem Cells by Cell Fusion

Speaker: Paul Feinstein, Associate Professor of Biology, Hunter College

Highlights

The generation of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) takes a long time, and often creates genetic abnormalities.

Feinstein is working on generating diploid iPSC by cell fusion. Feinstein’s lab has developed a fast and accurate screen for common

aneuploidies in ESC and iPSC using quantitative real time PCR.

Olfaction Dr. Feinstein’s lab is primarily focused not on stem cells and their generation, but on the surprisingly complex process of olfaction. Each olfactory neuron expresses only one unique olfactory receptor. But this does not come to pass like it does in the immune system, where there is genetic modification that leads to the clonal expansion of cell populations expressing unique antibodies. This is known from nuclear transfer experiments, in which the nucleus of an olfactory neuron was transferred into an oocyte to generate a whole mouse [1]. This mouse expressed the full complement of odorant receptors, not just the one from the neuron that spawned it. Its generation got Feinstein interested in the properties and generation of ESC and iPSC.

Cell Fusion

The first iPSC were made by reprogramming a diploid somatic cell with the four “Yamanaka factors” [2]. The resulting cells remained diploid, but the process takes weeks for murine cells and even longer for human cells. An alternate method is to fuse a diploid somatic cell with a diploid ES cell, generating iPSC within days. However, these cells are tetraploid, and therefore not particularly appropriate for either basic research or clinical purposes. Feinstein wanted to know: could we ablate the nucleus of the ESC before the cell fusion event in order to get a diploid iPSC, without killing the ESC in the process? And if we then ended up with diploid iPSC, how would we know which diploid nucleus was in the cell – that from the ESC or from the somatic cell? His lab thus developed an assay to determine if two cells have undergone a fusion event, and if so, which nucleus it contains.

They set up a system wherein the membrane of the somatic cell expresses a blue marker whose gene is flanked by loxP sites. The nucleus of the ES cell expresses a yellow marker, and these ES cells express the Cre recombinase. Thus a successful cell fusion event generating diploid iPSC containing nuclei from the somatic cell would yield cells with unlabeled (not yellow) nuclei and red membranes (after cre/lox recombination excised the blue membrane marker). This robust scoring system can be used to assay other cell fusion events, such as those used to make hybridomas. The Feinstein lab is hoping to patent it.

Detecting Aneuploidy

Feinstein’s work in screening for common aneuploidies in ESC and iPSC came directly out of these cell fusion experiments; he wanted to be sure there were no leftover chromosomes from the ablated ESC nuclei in their fused cells. Of course cells can be karyotyped, but that is expensive. And because reprogramming often introduces genetic

aberrations into ESC and iPSC, this type of screen is of utmost importance if these cells are ever to be used clinically.

Because olfaction cannot be studied in vitro, Feinstein had a lot of transgenic mice and thus many mouse ES cell lines - 141 of them, in fact - that had already been karyotyped. Of these cell lines, 73, or 51.8%, had abnormal karyotypes; and these were made in a core facility using standard procedures, indicating that many ESCs might share the same problem. Fifty three of these had numerical changes in chromosomes 8, 11, or Y (almost all ES cells are male). Feinstein’s idea was to use quantitative PCR to compare the amount of genetic material from these three chromosomes in a cell of unknown karyptype to that in a reference cell with a normal karyotype. Using this assay they recapitulated the results from their cell lines with known karyotypes, and they confirmed their results on new cell lines by having them karyotyped. They are therefore confident that it is robust enough to screen unknown cells.

Non-normal karyotypes can preclude ES cells from being used to inject blastocyts, and can prevent the production of germline mutations that is required for gene targeting and the generation of chimaeric mice. They would obviously also prevent cells from being used clinically. There is thus a real need for this fast, real-time PCR based screening for common anueploidies in ES and iPS cells that allows many cell lines to be analyzed within a day instead of the weeks required for conventional karyotyping of just a single clone.

References

[1] Li J, Ishii T, Feinstein P, Mombaerts P. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature. 2004 Mar 25; 428(6981):393-9. [2] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76. Epub 2006 Aug 10.

AFTERNOON SESSION 1: Applications and Implications

Stem Cell Research: Disparate Participation, Risks, and Benefits

Speaker: Harriet Washington, Award Winning Writer and Editor

Highlights:

Fear and rejection of medical research is not paranoia; it is a perfectly appropriate and adaptive response to historical realities.

The infamous Tuskegee syphilis study is only one of many studies in which a group was exploited in the name of medical research.

Informed consent is often anything but, and the requirement for it can be waived with shocking frequency.

Internal Review Boards (IRBs) are a step in the right direction, but should be comprised of 50% research subjects.

“A history that is rife with abuse and exploitation”

Harriet Washington, the author of Medical Apartheid: The Dark History of Medical Experimentation on Black Americans From Colonial Times to the Present and Deadly Monopolies: The Shocking Corporate Takeover of Life Itself, brought a dose of perspective and context in which to place the remarkable discoveries and innovations presented during the rest of the symposium. She said that stem cell research lies at “the intersection of our ethical and technological futures: what medicine should do and what medicine can do.” And that intersection, she noted, was the perfect place for people of conscience to be.

Ms. Washington pointed out that although the Tuskegee syphilis study is perhaps the most infamous case of a minority group being subject to medical experimentation without their consent or even their knowledge, it is a hardly a unique case. She spoke of the Havasupai, a tribe of Indians (their preferred term, she made sure to note) that lives in the Grand Canyon. The group has disproportionately high rates of type II diabetes, and was thrilled when researchers from Arizona State University approached them in 1989 to explore the genetic underpinnings of this diabetes risk. They were considerably less thrilled, however, when they learned that – without their knowledge or consent - the researchers in fact used their DNA to reveal an increased risk of schizophrenia as well as genealogical data that contradict their traditional stories. Ms. Washington cited this case to demonstrate that entire communities, and not just individuals, can be harmed by unendorsed medical experimentation.

The “powerless and voiceless” are often absent in ethical discussions, Ms. Washington said, and that does not include just minorities. She spoke of John Moore, a white man whose father was a doctor, who was treated at UCLA Medical Center for leukemia. But his physician, Dr. David W. Golde, realized that his cells were unique, and valuable; he patented them without Moore’s permission, and kept Moore coming back to the clinic to harvest more material by telling him he needed additional treatments. When Moore sued his doctor, The Supreme Court of California ruled that Moore had no property rights to his discarded cells or any profits made from them. But in terms of cell and tissue appropriation, as with other aspects of medical experimentation that she discussed, Ms. Washington said that “being legal doesn’t make it ethical.”

Ms. Washington said that distrust of the medical establishment is on the rise, among whites as well as blacks. She opined that this distrust is due partially to the untrustworthiness of the American medical system, and placed the responsibility of educating research subjects squarely on the shoulders of doctors and scientists, not on the subjects themselves. And she bemoaned the fact that this education is usually done at the hands of the very researchers conducting the study, who have a conflict of interest. She was quick to point out that she was not casting doubt on any researchers’ motivations, only noting that since the researchers conducting the study obviously believe in its worthiness it is only human nature for them to unconsciously overstate its benefits and downplay its risks.

“Informed” Consent?

Since the Nuremberg Trials in 1946, the United States has been a leader in requiring informed consent for participation in medical studies. But, Ms. Washington warned, that leadership is eroding. Already, she said, our country is a “crazy quilt” of risk, with consent for tissue collection being “presumed” in 28 states. So if you die in one of those states, the medical examiner can take your tissues and “transfer” them to brokers for a fee. Selling them, however, is illegal.

As of 1996, victims of trauma can be enrolled in experimental medical studies without their consent. One such study was the transfusion of an artificial blood substitute while it was still under development. Subsequent analysis demonstrated that people who received these products were subject to a higher risk of death than those receiving real blood, and the products are thus no longer administered.

Although anyone of any ethnicity can be the victim of a trauma, like a car accident, Ms. Washington points out that minorities are still overrepresented in such studies. Why? “Hospitals are located in predominantly black neighborhoods; they were built there for the express purpose of exploiting the local residents as research subjects, because it was thought that they would probably not ask too many questions or seek any recourse. Thus, many of the victims brought in to urban hospitals for trauma are black.“

To the criticism that one can’t very well ask informed consent of an unconscious trauma victim requiring an immediate blood transfusion, Ms. Washington counters that “in our zeal for better therapies and better treatment, we must remember that there are some studies that cannot ethically be done.”

Even if informed consent could always be attained by adequately educated subjects, the requirement for it can be waived. In 1990, the Department of Defense applied for, and received, a waiver of informed consent from the FDA. They wanted to test anthrax vaccines on their soldiers, and it was deemed too difficult and expensive to ask them all individually. So soldiers became another group forcibly enrolled in a medical trial without their knowledge. The waiver was overturned by a federal judge – but not until 2005. Moving Forward

Things are looking up, however. The flip side of forcing minorities to be research subjects without their knowledge or consent is barring them from studies that might benefit them. There were no Africans or African American samples in the Human Genome Project, which is somewhat ironic given that as the oldest human population Africans have the greatest amount of genetic diversity. But the Human Heredity and Health project currently being undertaken in Africa aims to remedy that exclusion, and

the Human Microbiome Project is comprised of 20% minorities. Already, ethnic differences in microbiomes have been uncovered.

The requirement for Internal Review Boards (IRBs) to monitor the ethics of research studies being done at any given institution is another positive development. Ms. Washington notes that these can (and have) been corrupted, which is unfortunate because people tend to remember headlines about these types of things going awry without thinking about all of the times that they go smoothly.

Ms. Washington noted that although it is not necessarily an accurate depiction of reality, African Americans view medical research as a “land of whites” that has nothing to do with them. Although the history of medical experiments on African Americans, and others, is somewhat gruesome, it is incumbent upon the medical community to fix the system so more subjects will be willing to consent to becoming study subjects to advance both medical knowledge and clinical treatments.

Hematopoietic Stem Cell Transplantation: The Search for the Perfect Donor

Speaker: Koen van Besien, Professor of Medicine, Weill Cornell Medical College Highlights

Bone marrow transplants have a long and successful history. Donors and recipients must be HLA matched. Umbilical cord blood does not require as close a match as adult bone marrow

transplants. A Brief History of Bone Marrow Transplantation

The field of stem cells as therapeutics holds immense promise, but is still largely in its infancy. Yet there is one area in which it has a tried and true history: treating hematopoietic disorders, especially leukemia.

Back in 1949, during the Manhattan Project, all sorts of studies were done with radiation. At that time, Leo Jacobson found that 1500rads of radiation would prevent a mouse from making blood and so the mouse would then die. But if the mouse’s spleen was removed from the mouse but left connected to it, and protected from the radiation, it could make new, healthy blood and the mouse would be ok. The existence of hematopoietic stem cells in the spleen was thus hypothesized, and the cells themselves were isolated only a few years later, in 1952. Bone marrow transplants were attempted in patients with leukemia in 1957, and the bone marrow did engraft, but the grafts all failed because of graft versus host disease.

Matchmaker, matchmaker, make me a match

In 1968 the Human Leukocyte Antigen (HLA) system was identified as the major determinants of graft rejection. Although this is a complex system with six polymorphic alleles, all six loci are found on chromosome six. Since everyone gets one copy of chromosome six – and all of the HLA types encoded by it – from each parent, siblings each have a 25% of matching each other. Five out of six alleles must match for a successful bone marrow transplant. The first successful transplants on patients with end stage acute myelocytic leukemia were performed in 1977, only 25 years after hematopoietic stem cells were first isolated, and the technique has been continuously optimized since then. Now, patients are first treated with only enough radiation to ablate all of their own hematopoietic stem cells while sparing the rest of their organs, then treated with immunosuppressants to combat graft versus host disease, and then HLA-matched bone marrow is transplanted in. Over one million such transplants have been performed worldwide.

Although the discovery of the HLA system enabled the development and proliferation of bone marrow transplant technology, its very existence is a severe limitation. Siblings have a one in four chance of matching, but not that many people – especially in the developed world, and certainly in China – have many siblings from which to choose. Africans in particular are more genetically diverse that other peoples, being an older population, and thus it is concomitantly more difficult for them to find matches. The establishment of international bone marrow registries has allowed people to find matches among unrelated strangers, and has saved numerous lives. Eight million potential bone marrow donors are currently enrolled in the registry in the US.

Cord Blood: Pluses and Minuses

The hematopoietic stem cells in umbilical cord blood are more immunologically naïve than those in adult bone marrow. They are also more tolerant, perhaps because the fetus is constantly being exposed to foreign maternal antigens that it must learn to tolerate. Thus, when transplanted, they do not need to match the recipient as well as adult cells do in order to avoid graft versus host disease. Four out of six, rather than five out of six, matching HLA alleles are usually sufficient. Moreover, a reduced rate of disease recurrence has been reported following umbilical cord blood transplantation compared to adult bone marrow transplantation – perhaps because of this very mismatching. Stem cells are present in a much higher concentration in cord blood than in adult blood marrow, and these stem cells have a greater proliferative capacity than their adult counterparts.

But – and this is a big but – there are not usually enough of these stem cells in a sample of cord blood for successful transplantation. Although they can grow faster than the adult cells, there are so few of them to start out with that they do not usually repopulate the transplant recipient’s blood for a few weeks. This leaves the recipient in a highly vulnerable, immunocompromised position. And because of the severity of their disease, many of these recipients do not have the few weeks to wait to expand a population of cord blood stem cells in vitro prior to transplantation.

Thus, cord blood can be transplanted, but along with adult cells that can proliferate rapidly, then disappear as the fetal cells take over [3]. Interestingly and importantly, the adult cells do not need to match the recipient. This new co-transplantation technology championed by Dr. Van Besien thus reaps the benefits of the mismatched, tolerant umbilical cord blood cells while obviating the slow and erratic hematopoietic recovery that used to accompany them. Although cord blood banking is still the province of private companies, hopefully, one day there will be lifesaving cord blood registries just as successful as the bone marrow registries have proven.

References

[1] Artz AS, van Besien K. Pre-transplant serum ferritin is prognostic but is it useful? Leuk Lymphoma. 2013 Jun; 54(6):1133-4. doi: 10.3109/10428194.2012.756483. Epub 2013 Jan 8. [2] van Besien K, Shore T, Cushing M. Peripheral-blood versus bone marrow stem cells. N Engl J Med. 2013 Jan 17; 368(3):287-8. doi: 10.1056/NEJMc1214025#SA1. [3] van Besien K, Liu H, Jain N, Stock W, Artz A. Umbilical cord blood transplantation supported by third-party donor cells: rationale, results, and applications. Biol Blood Marrow Transplant. 2013 May; 19(5):682-91. doi: 10.1016/j.bbmt.2012.11.001. Epub 2012 Nov 8.

http://marrow.org/Home.aspx

Using Stem Cells to Build an HIV-resistant Immune System

Speaker: Paula Cannon, Associate Professor, Molecular Microbiology & Immunology, Pediatrics, Biochemistry & Molecular Biology. USC Keck School of Medicine Highlights Although HIV can be controlled by drugs, treatment can be onerous so alternate

therapies are desired. The Berlin patient proved that HIV can be permanently cured by a transfusion of

CCR5-/- T cells. Zinc finger nucleases are one of a number of ways to disrupt genes in a targeted

manner. Human hematopoietic stem cells in which the CCR5 gene has been disrupted by

ZFNs render humanized mice resistant to HIV.

HAART Trouble

AIDS was first described in gay men in Los Angeles in 1981. For the rest of the eighties and a few years beyond, infection with HIV was tantamount to a death sentence. Now though, in this country at least, AIDS has become a terminal disease with which people can live for decades. The “game changer” came in 1995 with the introduction of a drug regimen known as Highly Active Anti-Retroviral Therapy, or HAART. AIDS patients who are on HAART are fine, and live normal lives.

That is, as long as they continue taking these drugs for their entire lifetimes. As soon as they stop taking the pills, the HIV that has been lying latent, integrated in their cells “like embers that can burst into flame,” rebounds. People might wish to stop taking these drugs because they are expensive (approximately half a million dollars over a normal lifespan) or because they are suffering side effects, but the virus still in their cells dictates that they cannot.

The Berlin Patient

Only one adult has thus far been cured of HIV, meaning that he had the virus and managed to get rid of it. He is an American named Timothy Ray Brown, but he is known as the Berlin Patient because luckily for him he was residing in Berlin in 2006, when he developed leukemia.

Most people at this point know that HIV requires the CD4 receptor to enter T cells. But it also requires a coreceptor, primarily CCR5. Brown’s doctor was aware that people who lacked the CCR5 receptor due to a genetic mutation were resistant to HIV infection, and this mutation did not seem to have any negative ramifications. Thus, when screening the extremely robust German Bone Marrow Registry for a match for Brown, he checked to see if any of the matches happened to be CCR5-/-. The mutation is relatively common in Western Europeans, and it turned out that one out of the 165 potential matches had it. After Brown received a bone marrow transplant from this CCR5-/- donor, he was found to be HIV free – and has remained so for the past five years. This served as a proof of principle that replacing an AIDS patients’ hematopoietic stem cells (HSC) with CCR5-/- HSC would cure them of AIDS. But a bone marrow transplant is a pretty big deal for someone who doesn’t feel like taking his pills anymore, and CCR5-/- donors might not always be so easy to find.

Zinc fingers are DNA binding domains that specifically recognize three nucleotides and are naturally found in transcription factors. A motif recognizing any three base pairs can be engineered and then grafted onto a restriction endonuclease that will cut at that

predefined DNA sequence. The generated double strand break provokes the cell’s DNA repair machinery to join the nonhomologous ends, disrupting the gene of interest. Cannon proposes to use zinc finger nucleases (ZFN) to disrupt the CCR5 gene in AIDS patients’ own HSC, rendering them CCR5-/- and thus resistant to HIV.

The protocol would be as follows: patients would receive G-CSF to get their HSC to migrate from the bone marrow to the blood so they can be easily isolated; then an mRNA encoding the ZFNs would be electroporated into the cells, where their transient presence is sufficient to induce just the “hit and run” gene therapy that is required without creating a risk of insertion into the genome like a viral vector might; and once CCR5 is knocked out, the cells are put back into the people. In contrast to a bone marrow transplant, the patient does not need to first undergo radiation to completely ablate all of their own HSC because they don’t all need to be replaced with the CCR5-/- versions for the therapy to be effective. Cannon chose to engineer hematopoietic stem cells, rather than mature T cells, because then all of their progeny would receive protection: T cells, macrophages, dendritic cells, and microglia. All of these cells can be targeted by HIV.

Cannon has already cured humanized mice of HIV this way [1], in yet another proof of principle. She used transient expression of ZFNs to disrupt CCR5 in human HSC isolated from umbilical cord blood, and then injected these cells into “humanized” mice – immunocompromised mice whose blood has been replaced with human blood, who are commonly used as models to study HIV and AIDS. Although only 17% of the HSC had their CCR5 successfully disrupted, this minority of cells was sufficient to render the mice resistant to HIV infection. This is partially because HIV actually selects for the propagation of the CCR5 cells by killing all of the other ones.

“Who gets to be the first patient?”

Of the 1.1 million Americans infected with HIV, only about a quarter of them have it well controlled by HAART. Those who also have lymphoma are obvious candidates to get CCR5-/- HSC because they must have their bone marrow ablated anyway, so this step – which must be undertaken in some form before the stem cell transplant - would not provide any additional risk for them. However, chemotherapy is so effective in this population now that the pool of HIV+ patients with lymphoma might actually be too small for a clinical trial.

Of the healthy HIV+ population, there are those who are on HAART but may desire to come off of it, and there are non-responders to HAART; their T cells do not rebound, even on when they loyally take their drugs. The risk/ benefit ration for this population is not quite as clear cut as it is for HIV+ people with lymphoma, however, since in order to receive the engineered HSC they would need to undergo some additional conditioning to reduce their unmodified HSC population.

References

[1] Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD, Holmes MC, Cannon PM. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol. 2010 Aug; 28(8):839-47. doi: 10.1038/nbt.1663. Epub 2010 Jul 2. [2] Li L, Krymskaya L, Wang J, Henley J, Rao A, Cao LF, Tran CA, Torres-Coronado M, Gardner A, Gonzalez N, Kim K, Liu PQ, Hofer U, Lopez E, Gregory PD, Liu Q, Holmes MC, Cannon PM, Zaia JA, Digiusto DL. Genomic Editing of the HIV-1 Coreceptor CCR5 in Adult Hematopoietic Stem and Progenitor Cells Using Zinc Finger Nucleases. Mol Ther. 2013 Jun;21(6):1259-69. doi: 10.1038/mt.2013.65. Epub 2013 Apr 16.

Locus Control Region Activity in T cells Derived in Vitro from Embryonic Stem Cells

Speaker: Benjamin Ortiz, Associate Professor of Biology, Hunter College

Highlights

Locus Control Regions (LCR) can convey copy number-dependent and cell-type specific transcription to a linked transgene in mice.

Thus far, transgenic mice have been required to recapitulate all of an LCR’s effects, making experimentation costly and time consuming.

The LCR must be present in the genome before differentiation. By putting a gene under the control of the TCR LCR into mouse embryonic stem cells, Ortiz was able to recapitulate all of its characteristics in T cells derived from those stem cells in vitro.

This system can be used to rapidly test LCR activity and translate it into tools achieving “robust therapeutic gene expression directed to the T cell progeny of the stem cell.”

Locus Control Regions

Locus Control Regions (LCR) are different from other cis-acting transcriptional regulatory DNA regions, like promoters and enhancers, in that they can confer both copy number dependence and lineage specific expression to a linked transgene in mice [2]. They can do this regardless of where they are inserted into the genome, as they have the ability to block any localized heterochromatin induced silencing effects. These features make them extremely valuable for the prospect of gene therapy.

T cells are to the immune system as a conductor is to an orchestra: they call the shots, and the other immune cells follow their orders. LCRs are not that common, but a number of them are found in gene loci active in T cells. Dr. Ortiz’s lab focuses on the LCR for the light, chain of the T cell receptor (TCR-). This LCR was discovered when researchers tried to make TCR-transgenic mice in the early 1990s – and couldn’t, until they also inserted the 13000 base pair region that contained what they retroactively deemed the LCR. The study of LCRs has remained largely dependent on transgenic mouse models, as their unique effects have proven refractory to recapitulation in a tissue culture system.

An In Vitro System

Two key insights helped Ortiz realize that inserting the TCR- LCR into mouse embryonic stem cells (mESC) might enable it to work as it did in whole mice. The first came from work with the -globin LCR, which indicated that for an LCR to work properly it could not be inserted directly into the genome of differentiated cells but must be present in the genome before cell lineage commitment takes place. The other was the development of a tissue culture system that allowed hematopoietic stem cells to differentiate along different paths. He used this system to show that when inserted into mESC, the TCR- LCR could drive the expression of a transgene in T cells but not in erythroid cells or monocytic cells derived from the same progenitor.

This in vitro system is capable of recapitulating all of the features of the LCR that make it so useful [1]. Examination of multiple clones demonstrated that expression of the transgene is independent of the integration site. qRT-PCR showed that the transgene is

expressed in a copy number dependent manner. FACS analysis revealed that the transgene is expressed with the same developmental timing during T cell maturation as that of the endogenous TCR- gene, and two mature T cell lines could not support these properties of the LCR. This cell culture system allows a given experiment examining LCR activity to be completed within six weeks, rather than the six months it can take in transgenic mice.

Tumor Targeting T cells

“The combination of stem cell technology and genetic engineering promises to eventually provide innovative therapeutic interventions for a wide variety of currently incurable diseases,” said Ortiz. He continued, “Some of these may require the insertion of a synthetic gene construct into the stem cell genome that encodes a new therapeutic gene product.” Harnessing the activity of LCRs will be essential to directing selective transcription of such gene products to the desired specific cell types, and an in vitro system is essential to accelerating the pace of progress towards a complete understanding of how LCRs work.

Altering the genome of a stem cell should affect all of its progeny. Sometimes that is desirable, but sometimes it is not. Using this system, T cells can be specifically engineered to produce a desired gene product – such as a receptor that can recognize, and generate an immune response against, tumor cells. The expression of such a receptor in all hematopoietic cells instead of just T cells could cause widespread inflammation – what Ortiz characterized as “the cellular equivalent of major drama.” Although such tumor targeting cells have already been made with mature T cells, manipulating stem cells is preferable as it would give patients a self-renewing supply of tumor targeting T cells.

References

[1] Lahiji A, Kucerová-Levisohn M, Lovett J, Holmes R, Zúñiga-Pflücker JC, Ortiz BD. Complete TCR-α Gene Locus Control Region Activity in T Cells Derived In Vitro from Embryonic Stem Cells. J Immunol. 2013. 191:472-9.

[2] Li Q, Peterson KR, Fang X, Stamatoyannopoulos G. Locus control regions. Blood. 2002 Nov 1; 100(9):3077-86.

AFTERNOON SESSION 2: Tissue Engineering and Regeneration The Developmental Biology of Regeneration

Speaker: Alejandro Sánchez Alvarado, Professor of Neurobiology and Anatomy, University of Utah

Highlights

Most multicellular organisms have stem cells. Planarians are non-parasitic flatworms that have been studied in depth since the

eighteenth century because of their remarkable regenerative capacity. The cells responsible for this regeneration are known as neoblasts. Planarian embryology is “weird,” and its study may precipitate a change in the

vocabulary we use to describe the development of all organisms.

“An ambassador to the invertebrate world”

In Madeline L’Engle’s novel, The Arm of the Starfish, Dr. Calvin O’Keefe has somehow managed to transfer the regeneration ability of starfish to humans, using it to repair major injuries. In the real world, Dr. Sanchez-Alvarado’s work deals with issues just as fundamental.

Sanchez-Alvarado is interested in learning the evolutionary origin of stem cells. What is their role in multi-cellular organisms? Since most multi-cellular organisms have stem cells, Alvarado does not need to use complex primate, mammalian, or even vertebrate cells as his model system. Like humans, planarians have bilateral symmetry along with all of the major developmental signaling pathways required to set it up. They develop through the same three germ layers as we do (endoderm, mesoderm, and ectoderm), and they have highly complex organ systems with many different cell types, just like we do. They have roughly 20,000 genes – again, like us. And they are famous, as literature dating back to the mid-1700s demonstrates that only a small fragment of tissue is capable of regenerating a complete worm. How small? In 1898, T.H. Morgan – the very same T.H. Morgan who popularized Drosophila melanogaster as a model system to study genetics [2] - found that 1/279 of the original animal could produce a whole new functional animal, with all of its concomitant tissue types, even germ cells [3]. Sanchez-Alvarado pointed out that that’s like cloning yourself by cutting off your finger and watching it grow into a complete new you.

Even before then, in 1893, undifferentiated cells were observed in planarians. They were deemed neoblasts. More modern microscopy has revealed that they look a lot like mouse embryonic stem cells, with decondensed chromatin and scant, highly basophilic cytoplasm. They are incredibly abundant, comprising about a quarter of all the cells in the planarian, and they are dispersed throughout the animal. They are self renewing, and although they are not all stem cells, they are the only somatic cells in the planarian capable of undergoing mitosis. They are almost constantly dividing, with roughly sixty percent of them cycling at any given time. They are selectively abrogated by ionizing radiation.

“Why put words in the embryo’s mouth? Why not let the embryos tell us their story in their own words?”

Although planarian development was described in detail in the 1890s, the mechanisms underlying it have still not been elucidated. And much more is known about the embryogenesis of other orders of flatworms than the freshwater planarians. Sanchez-Alvarado noted that, “the study of current model systems presupposes a number of conserved rules such as gastrulation and epiboly that are supposed to be necessary for normal embryonic development to occur.” When viewed with these predefined parameters in mind, he said, planarian development is “weird.” The “anarchic” behavior exhibited by the planarian blastomeres makes it difficult to understand how these cells form the three germ layers we know they do, how they communicate with one another, and ultimately how they organize into a coherent organism. Despite its weirdness, though, planarian development does share some analogies with mouse development. Sanchez-Alvarado wondered if these might be due to evolutionary conservation.

Alvarado wants to know the developmental history of the neoblasts. Recent unpublished work in his lab suggests that adult stem cell regulators are expressed in blastomeres, very early in development. However, a cohort of genes expressed in blastomeres is not detectable in neoblasts. These data suggest that neoblasts are a differentiated cell type that arises at a certain defined point during development. Mechanisms may be established during early embryogenesis to specify and perpetuate the neoblast fate, since these cells persist throughout the life of adult planarians. In order to more accurately define when and where during development neoblasts arise, Sanchez-Alvarado’s lab is working on culturing Schmidtea mediterranea (his planarian of choice) embryos in dishes and visualizing where and when in the blastomeres different cell populations appear.

Sanchez-Alvarado concluded by noting that the vocabulary used to describe embryology was coined well before any knowledge of genetics or cell biology, and is based on observing the development of only a very few of the Earth’s many species. He suggests that if the “weirdness” of planarian development is any indication of what other unstudied organisms experience, we may require a new vocabulary; in fact, rather than dictating to them the stages that they must pass through, we should sit back, observe, and “let the embryos tell us their story in their own words.”

References

[1] Guedelhoefer OC 4th, Sánchez Alvarado A. Amputation induces stem cell mobilization to sites of injury during planarian regeneration. Development. 2012 Oct; 139(19):3510-20. doi: 10.1242/dev.082099. Epub 2012 Aug 16.

[2] Sánchez Alvarado A. The freshwater planarian Schmidtea mediterranea: embryogenesis, stem cells and regeneration. Curr Opin Genet Dev. 2003 Aug; 13(4):438-44.

[3] Sánchez Alvarado A. Q&A: what is regeneration, and why look to planarians for answers? BMC Biol. 2012 Nov 8; 10:88. doi: 10.1186/1741-7007-10-88.

Two Short Examples of Engineering Cell Fate: Blood Stem Cell Therapy and Cardiac Drug Screening

Speaker: Peter Zandstra, Canada Research Chair in Stem Cell Bioengineering, Institute of Biomaterials and Biomedical Engineering, The Donnelly Centre for Cellular and Biomolecular Research,University of Toronto

Highlights

Umbilical cord blood has promise to treat blood disorders, but it is difficult to get enough cells.

Both in vivo and in vitro, hematopoietic stem cell number is controlled by the interplay of many factors and is inhibited by factors secreted by differentiated cells.

Computer modeling of these factors and their effects can help expand stem cell populations from cord blood in vitro, enabling their use in transplants.

Cardiac microtissues can be used to optimize tissue growth and perform high throughput drug testing.

Exploiting cell population dynamics to grow HSC

Hematopoietic stem cells (HSC) can be transplanted into patients to treat blood disorders, notably leukemias and lymphomas. Human bone marrow has been the traditional source for these cells, but it is often difficult to find an appropriate donor match for every patient. Umbilical cord blood is a preferable source of HSC, as it obviates the need for such an exact match. However, a successful transplant requires at least thirty million cells/ kg [2], and stores of umbilical cord blood often do not contain that many. Although these cells proliferate readily in vivo, getting them to do so in vitro has proven challenging. Thus, cost-effective, reproducible, timely, and scalable methods for expanding hematopoietic stem cells in cord blood would have immense clinical value.

Both in vivo and in vitro, the hematopoietic system maintains a complex balance of cell types from all of the different hematopoietic lineages. The stem cells mature, and as they do, differentiated cells secrete inhibitory factors that prevent the self-renewal of the stem cells that spawned them in a negative feedback loop. It is these very stem cells that are required for transplantation; thus an in vitro expansion system must prevent them from differentiating, but also counter the inhibitory signals emitted by the mature cells in the culture.

Strategies to expand HSC have traditionally tried to identify such inhibitory molecules one by one and counter each individually. Dr. Zandstra takes a more holistic, global approach. He appreciates that hematopoiesis is regulated by a system of intercellular communication, and that this entire system must be tweaked in order to effect any changes in hematopoiesis. Using in silico simulations, his lab has generated simple mathematical models of this complex system [4].

With these computer models, they have defined a culture system wherein adding fresh media to the culture on a daily basis sufficiently dilutes the stem cell inhibitory factors secreted by the mature cells [2]. Through this regulated feedback control, they can grow more cells for longer, at higher densities. By automating this system, they were able to expand a population of HSC in cord blood eighteen fold over the course of twelve days.

Cardiac microtissues for high-throughput screens

Cardiomyocytes have also been generated from stem cells, and it has been suggested that tissue grown from them be used as a “band aid” to replace damaged heart tissue in patients. However, engraftments of these tissues have not spurred a large clinical improvement. To optimize such “band aids,” Zandstra’s lab has made arrays of cardiac microtissues, embedded in three dimensional micropatterned matrices [1]. They can make roughly two hundred of these microtissues with a million cardiac cells, fewer than the number of cells in a typical neonatal rat’s heart. Traditional differentiation methodologies have not allowed for the measurement of the force generated by cardiomyocytes under different conditions, which is unfortunate since this force is the one of most important outputs required; engraftments often failed when the graft failed to beat with the heart into which it was placed.

Using the microtissue array, Zandstra can vary multiple parameters – cell composition, cell orientation, the composition and flexibility of the embedded matrix, electrical stimulation, and cyclic stretching – and assay their impacts on the frequency and length of contractions. In this way they hope to find the conditions that will turn stem cells into the optimal myocardial tissue for transplantation. They can also measure the force of contractions, electrical signal propagation, and changes in cardiac rhythm in the microtissues as they are exposed to different drugs to test the drugs’ safety and efficacy in a high throughput and combinatorial manner.

References

[1] Boudou T, Legant WR, Mu A, Borochin MA, Thavandiran N, Radisic M, Zandstra PW, Epstein JA, Margulies KB, Chen CS. A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. Tissue Eng Part A. 2012 May; 18(9-10):910-9. doi: 10.1089/ten.TEA.2011.0341. Epub 2012 Jan 4. [2] Csaszar E, Kirouac DC, Yu M, Wang W, Qiao W, Cooke MP, Boitano AE, Ito C, Zandstra PW. Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling. Cell Stem Cell. 2012 Feb 3; 10(2):218-29. doi: 10.1016/j.stem.2012.01.003.

[3] Csaszar E, Cohen S, Zandstra PW. Blood stem cell products: toward sustainable benchmarks for clinical translation. Bioessays. 2013 Mar; 35(3):201-10. doi: 10.1002/bies.201200118. Epub 2013 Jan 15. [4] Kirouac DC, Ito C, Csaszar E, Roch A, Yu M, Sykes EA, Bader GD, Zandstra PW. Dynamic interaction networks in a hierarchically organized tissue. Mol Syst Biol. 2010 Oct 5; 6: 417. doi:10.1038/msb.2010.71.

Engineering Human Tissues

Speaker: Gordana Vunjak-Novakovic, Professor and Vice-Chair, Department of Biomedical Engineering, Director, Laboratory for Stem Cells and Tissue Engineering, Columbia University

Highlights Tissue engineers can use stem cells to create the “spare parts” we may need as our

life spans extend. In vitro experiments must try to reconstruct in vivo environments in order to unlock

the full potential of stem cells. Scaffolds and bioreactors are as vital as cells in bioengineering these cell culture

environments. Defects in the bones of the head and face are prime candidates for bioengineering

because they vary in shape between individuals.

Stem cells: “the ultimate ‘tissue engineers’”

Dr. Vunjak-Novakovic started her presentation by pointing out that in ancient Greece, despite the populace’s dedication to physical fitness, life expectancy was in the range of thirty to thirty five years. But our eternal quest for the fountain of youth has had some success; humans are living longer than we ever have before. And we want to live better as well. But sometimes, selected tissues might need to be surgically removed, or they can become worn or diseased before the rest of the organism. Tissue engineers aim to make “spare parts” – much like those used in a car – to regenerate such tissues [5].

Tissue culture has been an invaluable aspect of laboratory research, but it has remained unchanged for roughly the past century and a half. According to Vunjak-Novakovic, although we “cannot possibly recapitulate” the in vivo environment in any built, in vitro system, we can certainly do better than continuing to grow cells in two dimensional Petri dishes. In order to more accurately mimic the physiological milieu, bioengineered cell culture environments must be contained within a three dimensional matrix, and they must expose cells to the appropriate hormones, cytokines, morphogens, and tissue specific transport and signaling molecules they would see in an in vivo setting. Moreover, cells grow differently in response to physical forces, like cell-cell and cell-ECM contact, motion, electricity, and stress, all of which are present in the body but have been lacking in standard tissue culture plates.

As an example of the importance of cellular context in in vitro systems, Vunjak-Novakovic pointed out that when cells were taken from primary tumors and put into tissue culture dishes, they lost their cancer signatures. When they were then placed into three dimensional models, they re-expressed the 600 genes that were upregulated in the tumors but not in the dishes. Thus these bioengineered systems can be valuable for basic research and drug discovery as well as for generating tissues for autologous transplantation.

Biomimetic Platforms

In order to get stem cells to realize their pluripotency in vivo – to actually turn into tissues that can be put into people who need them - they require a scaffold and a bioreactor. The scaffold is similar to that used to temporarily support a building; it provides structure, topology, and immobilized factors, much like the extracellular matrix (ECM) does in an in vivo setting, on which cells can be seeded and cultivated. Scaffolds are usually made of biodegradable materials that dissipate once the cells have grown appropriately.

A bioreactor further supports the growing cells in a three dimensional framework while exposing them to media at a determined flow rate that generates the appropriate concentration gradients of oxygen and various nutrients and other stimuli. Different bioreactors are made to mimic the specific conditions of different tissues and thus regulate tissue development. Studies with different microbioreactors have shown that human embryonic stem cells’ differentiation into cardiovascular cells is correlated with the level of hydrodynamic shear they experience and the transport rates of oxygen and metabolites around them [5], demonstrating that the “one size fits all” approach we have heretofore taken to tissue culture is not really viable for turning stem cells into organs we can use.

Putting a new face on things

Injuries to bones in the head and face, whether due to congenital defects, tumor removal, or trauma, have wide ranging psychological and social ramifications. These injuries are difficult to fix because the bones vary in shape from individual to individual [1]. This makes them attractive targets for bones engineered from a patient’s own stem cells.

Vunjak-Novakovic’s lab has successfully made a clinically sized, anatomically shaped, viable temporomandibular joint (TMJ) using human mesenchymal stem cells and a bioreactor scaffold-bioreactor system of their own design [3]. They chose to start with mesenchymal stem cells because they are readily easy to attain, being present in bone marrow and adipose tissue, and can differentiate into all of the cell types necessary for bone: cartilage, bone, adipose, and vascular tissue. However, Vunjak-Novakovic’s group has also successfully made bone substitutes out of human embryonic stem cells [4] as well as human induced pluripotent stem cells [2]. Experiments are currently under way to test these bone grafts in pigs, which are the standard model for head and face reconstruction since, believe it or not, they have the most similar facial shape and chewing mechanism to humans.

References

[1] Bhumiratana S, Vunjak-Novakovic G. Concise review: personalized human bone grafts for reconstructing head and face. Stem Cells Transl Med. 2012 Jan; 1(1):64-9. doi: 10.5966/sctm.2011-0020. Epub 2011 Dec 7. [2] de Peppo GM, Marcos-Campos I, Kahler DJ, Alsalman D, Shang L, Vunjak-Novakovic G, Marolt D. Engineering bone tissue substitutes from human induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2013 May 21; 110(21):8680-5. doi: 10.1073/pnas.1301190110. Epub 2013 May 7. [3] Grayson WL, Fröhlich M, Yeager K, Bhumiratana S, Chan ME, Cannizzaro C, Wan LQ, Liu XS, Guo XE, Vunjak-Novakovic G. Engineering anatomically shaped human bone grafts. Proc Natl Acad Sci U S A. 2010 Feb 23; 107(8):3299-304. doi: 10.1073/pnas.0905439106.

[4] Marolt D, Campos IM, Bhumiratana S, Koren A, Petridis P, Zhang G, Spitalnik PF, Grayson WL, Vunjak-Novakovic G. Engineering bone tissue from human embryonic stem cells. Proc Natl Acad Sci U S A. 2012 May 29; 109(22):8705-9. doi: 10.1073/pnas.1201830109. Epub 2012 May 14. [5] Vunjak-Novakovic G, Scadden DT. Biomimetic platforms for human stem cell research. Cell Stem Cell. 2011 Mar 4; 8(3):252-61. doi: 10.1016/j.stem.2011.02.014.

[6] http://magazine.columbia.edu/features/spring-2012/hearts-bones

© 2013 Center for Study of Gene Structure and Function. All rights reserved.

Media Christine Mummery Induced Pluripotent Stem Cells: The New Patient? Lorenz Studer hESC Derived Midbrain Dopamine Neurons for Cell Therapy in Parkinson’s Disease Paul Feinstein Generating Induced Pluripotent Stem Cells by Cell Fusion Koen van Besien Hematopoietic Stem Cell Transplation: The Search for the Perfect Donor Paul Cannon Using Stem Cells to Build an HIV-Resistant Immune System Benjamin Ortiz Locus Control Region Function in T Cells Derived in Vitro From Embryonic Stem Cells Alejandro Sánchez Alvarado The Developmental Biology of Regeneration Keynote Speakers James Thomson, Ph.D. Morgridge Institute for Research, University of Wisconsin Thomson graduated with a B.S. in biophysics from the University of Illinois in 1981. He entered the Veterinary Medical Scientist Training Program at the University of Pennsylvania, receiving his doctorate in veterinary medicine in 1985, and his doctorate in molecular biology in 1988. He derived the first human embryonic stem (ES) cell line in 1998 and derived human induced pluripotent stem (iPS) cells in 2007. He serves as Director of Regenerative Biology at the Morgridge Institute for Research in Madison, Wisconsin, is a professor in the Department of Cell and Regenerative Biology at the University of Wisconsin’s School of Medicine and Public Health and a professor in the Molecular, Cellular, and Developmental Biology Department at the University of California, Santa Barbara. He is also a founder and Chief Scientific Officer for Cellular Dynamics International, a Madison-based company producing derivatives of human induced pluripotent stem cells for drug discovery and toxicity testing. Harriet Washington Award-winning Medical Writer & Editor Harriet Washington is an award-winning medical writer and editor, and the author of the best-selling book, Medical Apartheid: The Dark History of Medical Experimentation on Black Americans from Colonial Times to the Present. In her work, she focuses mainly upon bioethics, history of medicine, African American health issues and the intersection of medicine, ethics and culture.

Medical Apartheid, the first social history of medical research with African Americans, was chosen as one of Publishers’ Weekly Best Books of 2006. The book also won the National Book Critics Circle Nonfiction Award, a PEN award, 2007 Gustavus Myers Award, and Nonfiction Award of the Black Caucus of the American Library Association. It has been praised in periodicals from the Washington Post and Newsweek to Psychiatric Services, the Economist, Social History of Medicine and the Times of London and it has been excerpted in the New York Academy of Sciences’ Update. Experts have praised its scholarship, accuracy and insights. Washington wrote Medical Apartheid while she was a Research Fellow in Ethics at Harvard Medical School. She has worked as a Page One editor for USA Today, as a science editor for metropolitan dailies and several national magazines, and her award-winning medical writing. Her work has appeared in Health, Emerge and Psychology Today, as well as such academic publications as the Harvard Public Health Review, the Harvard AIDS Review, Nature, The Journal of the American Medical Association, The American Journal of Public Health and the New England Journal of Medicine. Her awards include the Congressional Black Caucus Beacon of Light Award, two awards from the National Association of Black Journalists and a Unity Award from Emerge. She is the founding Editor of The Harvard Journal of Minority Public Health and has presented her work at universities in the U.S. and abroad. In her most recent book, Deadly Monopolies: The Shocking Corporate Takeover of Life Itself, Washington takes an in depth, eye-opening look at the 40,000+ patents on human genes and their harmful, even lethal, consequences on public health. Her other books include, Parkinson’s Disease, a monograph published by Harvard Health Publications, Living Healthy with Hepatitis C and she is co-author of Health and Healing for African Americans. Ms. Washington has taught at venues that include New School University, SUNY, the Rochester Institute of Technology, University of Rochester, Harvard School of Public Health and Tuskegee University. She has sat on the boards of many organizations, including The Young Women’s Christian Association, the School Health Advisory Board of the Monroe County Department of Health and the Journal of the National Medical Association, to name a few. Ms. Washington has also worked as a laboratory technician, as a medical social worker, as the manager of a poison-control center/suicide hotline, and has performed as an oboist and as a classical-music announcer for WXXI-FM, a PBS affiliate in Rochester, N.Y. She lives in New York City with her husband Ron DeBose. Dr. Alejandro Sánchez Alvarado, Ph.D. University of Utah Alejandro Sanchez Alvarado is an Investigator of the Stowers Institute for Medical Research and the Howard Hughes Medical Institute. He received his Bachelor’s Degree in Molecular Biology and Chemistry from Vanderbilt University in 1986, and his Ph.D. in 1992 in Pharmacology and Cell Biophysics at the University of Cincinnati School of Medicine, where he studied mouse embryonic stem cells and their in vitro differentiation under the tutelage of Dr. Jeffrey Robbins and Dr. Thomas Doetschman. In 1994, he joined the laboratory of Dr. Donald D. Brown at the Carnegie Institution of Washington, Department of Embryology as a postdoctoral fellow, and in 1995 was appointed Staff Associate. It was during this period that Dr. Sanchez Alvarado began to explore systems in which to molecularly dissect the problem of regeneration. In 2002, he became an Associate Professor and was promoted to full Professor in 2004 in the Department of Neurobiology and Anatomy at the University of Utah School of Medicine,

where he was later appointed H.A. & Edna Benning Presidential Professor (2010). Dr. Sanchez Alvarado has served in the National Advisory Council, National Institutes of General Medical Sciences, NIH (2008-2012), and is currently on the scientific advisory boards of the Mount Desert Island Biological Laboratory, Bar Harbor, MA; The Eugene Bell Center for Regenerative Biology and Tissue Engineering, Woods Hole, MA; the UCL Centre for Stem Cells and Regenerative Medicine, London, UK; the Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India; and the Latin American Society for Developmental Biology. He is also a Kavli Fellow of the National Academy of Sciences USA, and the recipient of a MERIT award from the National Institutes of Health and the E.E. Just Medal for Scientific Achievement, from the American Society for Cell Biology. He is also co-director of the Embryology course at the Marine Biological Laboratory in Woods Hole, MA. Dr. Sanchez Alvarado’s current research efforts are aimed at elucidating the molecular and cellular basis of animal regeneration using the free-living flatworm Schmidtea mediterranea.

Speakers Christine Mummery, Ph.D. Leiden University Medical Center, the Netherlands

Christine is Professor of Developmental Biology at Leiden University Medical Centre, The Netherlands and she is chair of the Department of Anatomy and Embryology. Her research has largely concerned mouse cardiovascular development and the role of growth factors. Over the last 10 years this has extended to the directed differentiation of mouse and human embryonic stem cells to cardiomyocytes and vascular cells. She pioneered studies characterizing cardiomyocytes from hES cells and was among the first to inject them in mouse heart and assess their effect on myocardial infarction. Her more recent work has concerned creating cardiac and vascular disease models based on induced pluripotent stem cells and their potential use in drug safety pharmacology and drug discovery. She has recently written a popular book on stem cells “Stem Cells: scientific facts and Fiction” (2011) intended as a semi-lay guide to stem cell biology and applications. She is also the founding editor in chief of Stem Cell Reports, the new journal of the ISSCR, editorial board member of Cell Stem Cells and Stem Cells and past president of the International Society of Differentiation (2010-2012). In 2010 she was elected as a member of the Royal Netherlands Academy of Arts and Science. In the same year she became a member of the board of the academy. She was recently awarded an ERC Advanced grant to continue research on cardiovascular derivatives of human pluripotent stem cells and their use as disease models. Lorenz Studer, M.D. Memorial Sloan-Kettering Cancer Center A native of Switzerland, Lorenz Studer graduated from medical school in 1991 and received his doctoral degree in neuroscience at the University of Bern in 1994. While there, he initiated studies with Christian Spenger, leading to the first clinical trial of fetal tissue transplantation for Parkinson’s disease in Switzerland. Studer next pursued his research interests at the National Institutes of Health (NIH) in Bethesda, Maryland, where he worked in the laboratory of Ron McKay. At the NIH he pioneered the derivation of dopamine cells from dividing precursor cells. In 1998, he was first to demonstrate that the transplantation of dopamine cells generated in culture improve behavioral symptoms in Parkinsonian rats.

In 2000, he moved to New York City where he started his research program at the Memorial Sloan- Kettering Cancer Center (MSKCC). Early contributions of his lab include the in vitro derivation of midbrain dopamine neurons from ES, nuclear transfer ES cells and parthenogenetic stem cells. His laboratory was also first to demonstrate “therapeutic cloning” in a mouse model of a CNS disorder, and he has pioneered studies on the directed differentiation, high-throughput screening and genetic modification of human ES cells. His most recent work increasingly focuses on the translational application of human pluripotent stem cells in disease modeling, drug discovery and cell therapy. He currently leads a large multidisciplinary consortium to pursue the first clinical application of human ES cell derived dopamine neurons for the treatment of Parkinson’s disease. He received numerous awards for his work including the Boyer Young Investigator award and, most recently, the Annemarie Opprecht Award. Studer is the Director of the Sloan-Kettering Center for Stem Cell Biology. He is a Member of the Developmental Biology Program and the Department of Neurosurgery at MSKCC and a Professor in Neuroscience at Weill-Cornell. Paul Feinstein, Ph.D.

Hunter College of the City University of New York Paul Feinstein, Ph.D. is Associate Professor of Biology at Hunter College. A graduate of the University of Pennsylvania in 1986, he obtained his Ph.D. in 1996 at Columbia University in molecular genetics and did his postdoctoral work at The Rockefeller University. Research in the Feinstein laboratory is focused on the generation of induced pluripotent stem cells (IPSCs), genetic manipulation, and mechanisms of olfactory function. For the last two decades, he and his colleagues have created hundreds of gene-targeted and transgenic mice for the study of how odorant receptors function in odor identification, neuronal maturation, axonal path finding, axonal identity and as a regulator of gene choice. During this time, he became an expert in the growth and manipulation of embryonic stem cells (ESCs). Dr. Feinstein was co-author on the first cloning of a mouse from the postmitotic nucleus of a (olfactory) neuron by nuclear transfer (nt) into oocytes, followed by generation of ntESCs and, subsequently, the production of a live animal with ntESCs injected into tetraploid chimeras. His interest in generation of IPSCs is derived from an experiment published by another group whereby ESCs fused with somatic cells lead to rapid reprogramming of somatic nuclei and the generation of tetraploid ESCs. Koen van Besien , M.D. Weill Cornell Medical College Koen van Besien serves as the Director of the Stem Cell Transplant program at Weill Cornell Medical Center and New York Presbyterian Hospital. He received his undergraduate degree from the Facultes Universitaires at Namur and his medical degree from the University of Leuven, both in Belgium. After postgraduate training in Belgium and at Indiana University, he became a member of the transplant program at MD Anderson Cancer Center in Houston. He was the director of the transplant and lymphoma programs at the University of Chicago from 2001 until 2011. Dr. van Besien’s research interests include bone marrow transplantation and lymphoma treatments. He has most recently focused on the development of novel transplant conditioning regimens and on improving umbilical cord blood transplantation. He is the author or coauthor of over 200 publications. He is editor in Chief of Leukemia and

Lymphoma, a member of the editorial boards of Biology of Blood and Marrow Transplantation and Bone Marrow Transplantation, and a frequent reviewer for other journals. He is an active clinician and is board certified in Internal Medicine, Hematology and Oncology. Paula Cannon, Ph.D. University of Southern California Keck School of Medicine Paula Cannon PhD is an Associate Professor of Microbiology at the Keck School of Medicine at the University of Southern California, where she leads a research team that studies viruses, stem cells and gene therapy. She obtained her PhD from the University of Liverpool in the United Kingdom, and received post-doctoral training as an HIV scientist at both Oxford and Harvard Universities. Although HIV remains the main focus of her work, she also studies highly pathogenic hemorrhagic fever viruses, including Ebola and Lassa fever viruses. Dr. Cannon has a long-standing interest in the development of gene therapy as a clinical approach to treating HIV infection and her recent work in this area is aimed at disrupting the viral co-receptor, CCR5, using zinc finger nucleases (ZFNs). This approach is being evaluated in human hematopoietic stem cells, to address whether such a therapy could result in a ‘functional cure’ for AIDS patients. Dr. Cannon’s research is funded by both the National Institutes of Health and the California Institute for Regenerative Medicine.ch for the Benjamin Ortiz, Ph.D. Hunter College of the City University of New York Benjamin Ortiz, Ph.D. is Associate Professor of Biology at Hunter College. He pursues research on gene regulation during T cell development, while fostering the professional development of numerous members of Hunter’s diverse and talented student body. His laboratory has trained a diverse array of Hunter College alumni who are now pursuing biomedical research careers across the country. His lab studies a Locus Control Region (LCR), a DNA segment harboring potent gene regulatory activity in the T cells of the immune system. His lab has most recently pioneered the study of LCR activity in T cells derived in vitro from embryonic stem cells. This breakthrough promises to speed the translation of basic research on LCR activity to the design of gene therapy strategies against diseases such as cancer, inherited immunodeficiencies and AIDS. Dr. Ortiz is a Brooklyn native and product of the NYC public schools. He received his B.A. in Biology at Hunter College. The Minority Access to Research Careers (MARC) program at Hunter supported his first research experience, obtained in the laboratory of Dr. Robert Dottin. He was awarded a Howard Hughes Medical Institute Predoctoral Fellowship, with which he went on to earn his Ph.D. in Immunology from Stanford University working in the laboratory of Dr. Alan Krensky. He then conducted postdoctoral research at the University of California, Berkeley in the laboratory of Dr. Astar Winoto. Dr. Ortiz has been on the Hunter College Faculty since 2000. His research has been awarded several grants including a National Science Foundation (NSF) CAREER award, and SCORE and “R01” grants from the National Institutes of Health (NIH). He was also among the first recipients of individual investigator research grants from the Empire State Stem Cell Research Program (NYSTEM). He has served on grant review panels for the Department of Defense, NSF and NIH.

Peter Zandstra, Ph.D. Centre for the Commercializationof Regenerative Medicine, University of Toronto Dr. Zandstra is a Professor in the Institute of Biomaterials and Biomedical Engineering, the Department of Chemical Engineering and Applied Chemistry, and the Donnelly Centre at the University of Toronto. He is also a member of the McEwen Centre for Regenerative Medicine and the Heart and Stroke/Richard Lewar Centre of Excellence. He currently acts as Chief Scientific Officer for the Centre for the Commercialization of Regenerative Medicine (www.CCRM.ca). Research in the Zandstra Laboratory is focused on the generation of functional tissue from adult and pluripotent stem cells. His groups’ quantitative, bioengineering-based approach strives to gain new insight into the fundamental mechanisms that control stem cell fate and to develop robust technologies for the use of stem cells and their derivatives to treat disease. Specific areas of research focus include blood stem cell expansion and the generation of cardiac tissue and endoderm progenitors from pluripotent stem cells. Dr Zandstra’s accomplishments have been recognized by a number of awards and accolades including a Guggenheim Fellowship and the McLean Award. Dr Zandstra’s strong commitment to training the next generation of researchers is evidenced by his role as the Director of the undergraduate Bioengineering Program. Gordana Vunjak-Novakovic Ph.D. Columbia University Gordana Vunjak-Novakovic is the Mikati Foundation Professor of Biomedical Engineering, and a Professor of Medical Sciences at Columbia University in New York. She directs the Laboratory for Stem Cells and Tissue Engineering, the Stem Cell Imaging Core, the Bioreactor Core of the national Tissue Engineering Center, and the Stem Cell Core at Columbia University. She is the lead for bioengineering for the Columbia Stem Cell Initiative, and serving on the Board of Directors of the Center for Advancement of Science in Space. The focus of her research is on engineering functional human tissues using stem cells, biomaterials, and bioreactors, for regenerative medicine and study of development and disease. She has extensively published and cited (320 papers, 12,000 citations), has 62 patents, and gave 260 invited lectures. Gordana is a frequent advisor to government and industry, a distinguished editor for NIH, a member of editorial boards of 16 scientific journals, and a member of the National Academy of Engineering of the United States.