Hereby you receive the final program for our 3 rd
Waddensymposium organized by the
Department of Immunohematology and Blood Transfusion of the LUMC.
Location:
Grand Hotel Opduin
Ruijslaan 22
1796 AD De Koog (Texel)
The Netherlands
Department of Immunohematology and Blood Transfusion
Symposium secretariat: Amber Günthardt
Mail: a.n.gunthardt@lumc.nl
Phone: +31 71 526 38 27 cell phone : +31.6.53527185
Organizing committee:
Willem Fibbe
Frank Staal
Christine Mummery
Gijs van den Brink
Melissa van Pel
Jaap Jan Zwaginga cell phone +31.6.50655116

18.00
20.00
Sunday 27 th of June
Final program, 27 th of June till the 30 th of June 2010
Arrival Hotel Opduin, Texel
Dinner

Monday 28th of June
07.30 - 08.15 Breakfast
08.15 - 08.30 Welcome, symposium in perspective: Willem Fibbe, Leiden [The Netherlands]
Gene therapy and HSC
Chair: Jaap Jan Zwaginga
08.30 – 09.05 Stefan Karlsson [Sweden]
“Pathogenesis of Diamond-Blackfan anemia: Implications for future therapeutic strategies”
09.05 – 09.40 Elaine Dzierzak [the Netherlands]
“Of mice, men, and hematopoietic stem cells”
09.40 – 10.15 Frank Staal [the Netherlands]
“Wnt signaling strength regulates developmental checkpoints in hematopoiesis”
10.15 – 10.50 Salima Hacein-Bey-Abina [France]
“Gene Therapy clinical trials ongoing at Necker Enfants Malades Hospital:
An overview”
10.50 – 11.30 Coffee break
11.30 – 11.50 Frits Koning [the Netherlands]
“A novel lymphocyte precursor in the intestine”
11.50 – 12.50 Abstracts
Kenichi Miharada [Sweden] “ Cripto selectively expands a distinct population..”
Marinus Nauw [the Netherlands] “ Treatment strategies and molecular…”
Cheryl Dambrot [the Netherlands] “ Optimization of reprogramming of induced...”
Karolina Portalska [the Netherlands] “ Senescence of Human mesenchymal…”
13.00 – 13.45 Lunch
14.00 – 18.00 Social event (transfer at 14.00 hours)
18.00 – 20.00 Diner
Chair: Frank Staal
20.00 – 21.30 Chris Baum [USA]
“Genetic modification of hematopoietic cells – challenges and prospects”
Adrian Thrasher [UK
“Gene Therapy for Inherited Immunodeficiency: successes and future challenges”

Tuesday 29 th of June
07.30 – 08.30 Breakfast
Stem cell niche
Chair: Gijs van den Brink
08.30 – 09.05 Gijs van den Brink [the Netherlands]
“Negative feedback signaling in intestinal epithelial homeostasis"
09.05 – 09.40 Eduardo Moreno [Spain]
”The competitive nature of stem cells”
09.40 – 10.15 Kristel Kemper [the Netherlands]
“Colon cancer stem cells: isolation & identification”
10.15 – 10.45
Coffee break
10.45 – 11.20 Simon Mendez-Ferrer [USA]
“Nestin+ MSCs: self-renewing stem cells with HSC niche functions”
11.20 – 11.55 Tsvee Lapidot [Israel]
“Dynamic Interactions Between the Nervous and Immune Systems with
the Microenvironment, Regulate Normal and Leukemic Human Stem Cells”
11.55 - 12.30 Kateri Moore [USA]
“Model Systems to Elucidate Molecular Mechanisms in Stem Cell Niches”
12.30 – 13.00 Stephane Corbel [USA]
“Substrate Elasticity Regulates Skeletal Muscle Stem Cell Self Renewal in Culture”
13.00 – 13.30 Lunch
13.45 – 18.00 Social event (transfer at 13.45 hours)
18.00 – 20.00 Diner
Chair: Willem Fibbe
20.00 – 21.30 Ihor Lemischka [USA]
“ Pursuing Pluripotency”

Wednesday 30 th of June
07.30 – 08.30 Breakfast iPS
Chair: Christine Mummery
08.30 – 09.00 Uli Martin [Germany]
“Generation of human iPS cells from cord blood, long term expansion in
suspension culture and differentiation into functional cardiomyocytes”
09.00 – 09.30 Chad Cowan [USA]
“
Programming and Reprogramming: New Approaches for Understanding
Disease”
09.30 – 10.00 Toshio Suda [Japan]
“In vitro Acquisition of Pluripotency in Primordial Germ Cells”
10.00 – 10.45 Abstracts
Catherine Robin [USA] “ Live imaging of the first hematopoietic stem…”
Sunita D'souza [USA] “ Diverse stem cell projects supported by the nystem…”
Harald Mikkers [the Netherlands] “Non-coding RNAs: guardians of development?”
10.45 – 11.00
Coffee break
11.00 – 11.45 Abstracts
14.00
Christoph Schaniel [USA ]
“Induced Pluripotent Stem Cells as Models of Diseases”
Christian Freund [the Netherlands]
Richard Davis [the Netherlands]
11.45 – 13.00
Position paper
13.00 – 13.30 Lunch
13.35
Departure by bus to Harbour
Departure boat to Den Helder
“Derivation of iPS cells from patients with… “
“Mouse pluripotent stem cell models of a …”
END OF MEETING

ABSTRACTS

MONDAY
Pathogenesis of Diamond Blackfan anemia: Ribosomes and Regulation of
Hematopoiesis
Pekka Jaako, Johan Flygare, Karin Olsson, Axel Schambach, Christopher Baum, Jonas Larsson,
David Bryder, Stefan Karlsson
Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Presently, many different DBA disease genes are known that all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. In addition, several other hematological disorders have a defect in ribosomal function, for example Schwachman-Diamond Syndrome and Dyskeratosis
Congenita and most recently, haploinsufficiency of Ribosomal Protein S14 was identified as an important genetic defect contributing to the pathogenesis of the 5q- syndrome. In DBA, the ribosomal protein S19 (RPS19) gene is the most frequently mutated (25 % of the patients). In order to study pathophysiology and to evaluate novel therapies, we generated a mouse model with
RPS19 deficiency. Since RNA interference -mediated RPS19 down regulation has been shown to result in a DBA phenotype in human cells in vitro, we decided to use the short hairpin RNA
(shRNA) technology to create an RPS19-deficient mouse model for DBA. We designed miR30 styled shRNAs against RPS19 and introduced them into mouse embryonic stem (ES) cells downstream of the collagen A1 locus using site-specific recombination. The resulting ES cell clones contain a single RPS19-targeting shRNA under the control of a doxycycline-responsive promoter. We have generated and characterized two mouse models expressing different RPS19targeting shRNAs (shRNA-B and shRNA-D). In general, this system allows an inducible and dose-dependent regulation of shRNA expression providing an ideal tool to study conditions like
DBA that are caused by haploinsufficient expression of a protein. Doxycycline administration led to around 50 % reduction in RPS19 protein in whole bone marrow. RPS19-deficient mice showed a modest reduction in erythrocyte number and gradual elevation in mean corpuscular volume
(MCV). In contrast to DBA patients, RPS19-deficient mice showed a severe reduction in white blood cell number, and after early compensation, also in platelet number. After 10 days of doxycycline administration, FACS analysis of bone marrow revealed a general increase in hematopoietic stem and progenitor numbers, which is likely to reflect the attempt to compensate for RPS19-deficieny. At the same time-point, we observed a decrease in the number of proerythroblasts and late erythroblasts, while the levels of erythroid progenitors were comparable between RPS19-deficient and control mice. Proliferative potential of erythroid progenitors was evaluated at a clonal level in vitro. When MegE or CFU-E progenitors from induced mice were cultured in presence of doxycycline, the number and size of the erythroid clones were decreased compared to controls. However, when RPS19 expression was restored by culturing the cells without doxycycline, the observed proliferation defect of MegE clones was completely restored while the rescue of CFU-E clones was only partial. We also noticed that RPS19-deficient megakaryocytes appeared smaller in size compared to controls. Importantly, transduction of
RPS19-deficient cells with a lentiviral vector overexpressing sequence-modified RPS19 cDNA rescued the proliferation and colony-forming defects in vitro demonstrating that the erythroid phenotype is specifically due to down regulation of RPS19. In summary, we have generated two novel mouse models for RPS19-deficient DBA that recapitulate the key erythroid phenotype seen in patients based on both FACS analysis and single-cell proliferation assays. The location of the erythroid defect has been identified at the transition of the CFU-E/Proerythroblast transition.
These models will serve as a good tool to determine the molecular mechanisms responsible for
DBA and also to test gene replacement therapies.

MONDAY
Wnt signaling regulates critical steps in hematopoiesis and lymphopoiesis
Frank J.T. Staal
The Wnt signaling pathway has been implicated in regulation of haematopoiesis through a plethora of studies from many different laboratories. However, different inducible gain- and loss-of-function approaches retrieved controversial and some times contradictory results. Different levels of activation of the pathway, dosages of Wnt signaling required and the interference by other signals in the context of Wnt activation collectively explain these controversies. Gain-of-function or in vitro exposure to WNT proteins and more specifically WNT3a was shown to enhance hematopoietic stem cell (HSC) activity but its exact role was still not completely understood. In a recent study we analyzed the hematopoietic system of mice deficient for this specific Wnt gene. Wnt3a deficiency results in early embryonic lethality around embryonic day 12.5 (E12.5), precluding analyis in adult mice, but allowing hamatopoiesis to be studied in fetal liver and in the just colonized thymic rudiment. Notably, we showed that long-term HSCs and multipotent progenitors are reduced in fetal liver in situ and have severely reduced longterm reconstitution capacity as observed in serial transplantation assays. Of interest, deficiency in Wnt3a leads to complete abolishment of canonical Wnt signaling in fetal liver hematopoietic stem and progenitor cells. This HSC deficiency is not explained by altered cell cycle or survival and is irreversible since it cannot be restored by transplantation into Wnt3a competent mice. In addition, Wnt3a deficiency differentially affects myeloid and B-lymphoid lineages with myeloid cells being affected at the progenitor level, while B lymphopoiesis is apparently unaffected. Immature thymocytes however were reduced in cell numbers due to lack of Wnt3a production by the thymic micro environment. Our results show that while in the thymus Wnt3a provides cytokinelike, proliferative stimuli to developing thymocytes, Wnt3a regulates cell fate decisions of fetal liver HSC in a non-redundant way.
These results combined with published work by other investigators indicates that absence of Wnt signaling and very high Wnt signaling both are detrimental to HSC self renewal, suggesting an optimum Wnt signaling strength somewhere in between. Thus, these results show the importance of the correct dosage of WNT signals for HSC biology. However, this concept has not been tested by experiments yet. Here we make use of several unique mouse models that correspond to different levels of Wnt signaling. These mice comprise of a floxed APC allele that deletes exon 15 on one chromosome, combined with different point mutations on the other allele. The heterozygous lox15/wt mice have 2 fold increased Wnt signaling, whereas the 1572T and 1628N have 5 and 10 fold higher than wt Wnt signaling as measured by expression of universal the Wnt target gene, Axin2.
The mice with both alleles deleted have 30-40 fold higher Wnt signaling strength.
Deletion of the floxed allele is brought about by a Cre-GFP MSCV retrovirus. Our data show that there is a lineage specific optimum Wnt signaling strength, namely from low
(HSC, 2 fold), intermediate (myeloid, 5 fold) to high (thymocytes , 10 fold) Wnt signaling dosage. This suggests that Wnt signaling strength can regulate self renewal vs. differentiation decisions as well as lineage fate decisions during hematopoiesis.

MONDAY
A novel lymphocyte precursor in the human small intestine
Jennifer Tjon, Frederike Schmitz, Sara de Boo, Marco Schreurs, Chris Mulder, Ton
Langerak, Tom Cupedo, Jeroen van Bergen and Frits Koning
Celiac disease (CD) patients are intolerant to gluten and most patients respond well to a gluten free diet. In a subset of patients, however, symptoms remain despite a gluten free diet. In such refractory CD (RCD) patients an aberrant monoclonal lymphocyte population is present in the intestine that can develop into lymphoma, a usually fatal complication. These aberrant cells are characteristically surface T cell receptor (TCR)-
CD3 negative but do express CD3 intracellular. It is currently unknown from which cell type these aberrant cells originate. To determine the origin of these cells we isolated cell lines from small intestinal biopsies of RCD patients that bear the characteristic surface
TCR/CD3- phenotype. We have carried out an extensive analysis of cell surface expressed markers as well as microarray analysis and found that these cells display a unique phenotype characterized by the lack of lineage markers and distinct to that of classical NK or T cell precursors and lymphoid tissue inducer cells. Strikingly the cells express both markers that are associated with early T cell development as well as late NK cell development. Based on the surface phenotype of the cell lines a multicolor FACSstaining protocol was developed to determine if these cells also exist in the intestine of healthy individuals and uncomplicated CD. Such cells could be readily identified in the intestine of adults but not in the fetal intestine. Together these results demonstrate that in the intestine a previously unrecognized lymphocyte precursor is present that is not yet fully committed to either the T of NK cell lineage. This may be indicative for extrathymal
T cell development. As the aberrant cells in RCD always display the same phenotype, this indicates that the cells from which they derive are prone to monoclonal outgrowth and malignant transformation, potentially due to the inflammatory environment induced by gluten intake.

MONDAY
Cripto selectively expands a distinct population of hematopoietic stem cells expressing the cell surface receptor GRP78.
Kenichi Miharada , Göran Karlsson, Jonas Larsson, Emma Larsson, Kavitha Siva, and
Stefan Karlsson
Cripto is a member of EGF-CFC family and has been identified as an important factor for the proliferation/self-renewal of ES and tumor cells. The role for Cripto in the regulation of hematopoietic cells is unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro . The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL HSC had higher expression levels than purified hematopoietic progenitors. After two weeks culture in serum free media supplemented with SCF, TPO and recombinant Cripto,
CD34-KSL cells formed a large number of colonies, including GEMM colonies, while control cultures without Cripto generated few colonies and no GEMM colonies. CD34-
KSL cells were cultured with or without Cripto for 2 weeks and transplanted to lethally irradiated mice. Cripto treated donor cells showed higher chimerism (p=0.0031) than cells cultured without Cripto. In order to define the target population and the mechanism of
Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion mediated by Cripto whereas GRP78- HSC did not respond. In contrast, all lineage negative cells were Glypican-1 positive. We therefore sorted these two
GRP78+CD34-KSL and GRP78-CD34-KSL populations and transplanted to irradiated mice after culture with or without Cripto. Interestingly, fresh GRP78-CD34-KSL cells showed higher chimerism than GRP78+CD34-KSL cells. However, Cripto selectively expanded GRP78+CD34-KSL but not GRP78- HSC after 2 weeks of culture as indicated by a higher level of chimerism than freshly transplanted cells. These data strongly suggest that Cripto is a potent factor for expansion of a distinct population of GRP78 expressing
HSC.

MONDAY
Treatment strategies and molecular mechanisms underlying endoglin related vascular defects
R.M.J.P. Nauw , S. van den Brink, C.J. Westermann, H-J. Mager, F. Disch, R. Snijder,
F. Lebrin, C.L. Mummery
Background
Mutations in Endoglin cause HHT1. Absence of Endoglin severely disrupts vascular smooth muscle cell (VSMC) / endothelial cell (EC) association, resulting in weak vessel walls, severe nosebleeds and muc o taneous telangiectases. Recently, our clinical collaborators discovered that Thalidomide reduced nosebleeds dramatically in some HHT patients. We are investigating the underlying mechanisms using mutant mouse embryonic stem cells (mESC) and deriving induced pluripotent stem (iPS) cells as models in humans.
Design and methods
A collagen based assay allows induction of vasculogenesis and angiogenesis mESC in response to FGF and VEGF. Adult mouse and human skin fibroblasts can now be reprogrammed into iPS cells that resemble ESCs. hiPS cells from HHT1 patients are being developed as models to identify molecular mechanisms of VSMC/EC association and elucidate Thalidomide action.
Results
Thalidomide was shown to induce EC/VSMC association in differentiated vascular derivatives of Eng
+/-
mESC. Since Eng
-/-
embryos die in utero we derived iPS cells from
(Eng mutant) mouse embryos with view to deriving lines lacking the gene entirely (Eng
-/-
). We now have Eng
+/-
miPS cell lines with Eng
-/-
pending. In addition, we have derived fibroblasts from waste tissue from control individuals and HHT1 and HHT2 patients. Cell lines are growing and are presently being characterized for their ability to form ECs and
VSMCs in culture. Analysis of the effects of Thalidomide will follow with view to determining whether miPS and their human counterparts show similar responses.
This work is supported by NHF grant 2008B106 and SWORO.

MONDAY
Optimization of reprogramming of induced pluripotent stem cells (iPS) for use with patient material
Cheryl Dambrot , Christian Freund, Dorien Ward-van Oostwaard, Douwe Atsma,
Christine Mummery
Human Embryonic stem cells (hESC) are pluripotent and can self-renew indefinitely in culture. Since they can be differentiated into derivatives of the three germ layers they have been a useful tool in studying developmental processes, for drug and toxicology testing and as disease models. However, the derivation of embryonic stem cells from surplus early human embryos is banned in many countries due to ethical reasons. The derivation of induced pluripotent stem (iPS) cells in 2006 has opened a new area of stem cell research, particularly in regards to disease modeling. However, efficiencies are still low and the search for an optimal source of patient material is ongoing. Here we attempted to optimize reprogramming using different viral systems and investigated various cell types for later use with disease specific patient material. Primary skin fibroblasts were isolated from patient biopsies as small as 4mm and expanded. Two isolation methods were compared: Either outgrowth from small skin pieces or using an enzymatic collagenase digestion. Over a period of 5 days our results showed a 2 to 2.5 fold increase in the number of fibroblast obtained using enzymatic digestion compared to the outgrowth method. In order to test alternative sources for reprogramming, cells were also grown from oral mucosa using the fibroblast outgrowth method or T-cells were isolated from blood using a Ficoll separation. Oral mucosa cells were reported to endogenously express two of the transcription factors needed for reprogramming
(OCT3/4 and SOX-2). Using the 4 Yamanaka factors in retroviral vectors neither cells from oral mucosa or T-cells could be reprogrammed. By contrast skin fibroblasts were successfully reprogrammed. The iPS cells expressed the typical set of markers present in hESC, such as OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA1-60 and TRA1-81.
Following an embryoid body formation iPS cells differentiated into derivatives of the three germ layers, such as neurons (expressing βIII-tubulin), smooth muscle actin cells (α-
SMA), and endoderm (αAFP). Other sources currently under investigation include keratinocytes, for their easy accessibly from hair and peripheral blood endothelial progenitor cells.
Compared to retroviral vectors, lentivirial vectors have the advantage of large insertion size, allowing a single cassette to contain all four factors as well as an inducible factor. In addition the insertion of a LoxP site into the lentiviral vector allows removal of the transgenes, providing a number of benefits. We have successfully reprogrammed fibroblast using both viral methods.

MONDAY
Senescence of Human Mesenchymal Stromal Cells. The Influence of Ageing on
Endothelial Differentiation.
Karolina Janeczek Portalska, Hugo Fernandes, Clemens van Blitterswijk, Jan de Boer.
Human mesenchymal stromal cells (hMSCs) are adult stem cells which can be isolated from the bone marrow, expanded in the culture dish and differentiated into various cell types. Therefore hMSCs are increasingly used in regenerative medicine as a source of cells for restoring worn-out or damaged tissues such as cartilage, cardiac muscle or bone.
Since the amount of MSCs in the bone marrow is low, expansion is required prior to laboratory or clinical use. In this report we evaluate the influence of prolonged expansion on the ability of hMSCs to differentiate towards the endothelial lineage and compare it with adipogenic and osteogenic differentiation.
We cultured hMSCs on tissue culture treated plastic until reaching up to 80-90% confluency and then reseeded them at 5000 cell per cm
2
for further expansion. After every such step cells were frozen for further study. We created a bank of cells of 10 consecutive passages which were then used in differentiation assays. During the expansion phase cells beyond passage 6 exhibited morphological abnormalities (irregular, star-like shape and general cell enlargement). Also there was debris present in the medium. For adipogenic differentiation cells were seeded at the density 5000 per cm 2 in adipogenic medium and cultured for 21 days. Then Oil Red O staining was performed.
Ability to undergo osteogenic differentiation was assessed by comparing the activity of alkaline phosphatase in cells cultured with medium containing dexamethasone. This activity was measured with a CDP Star assay. To obtain endothelial differentiation, cells were cultured in EGM-2 medium (Lonza) for 10 days and then seeded on Matrigel. After
24 hours capillary-like structure formation was quantified. After a further 10 days of culture on Matrigel, cells were recovered and stained for endothelial markers.
As expected, with increasing passage number hMSCs proliferate slower than cells from early passages. Cells from passage 10 stopped proliferating at all and started to die. Our results suggest that hMSCs start to loose their multipotency almost immediately from the moment of in vitro culturing. Concomitant with increasing passage number the efficiency of hMSCs differentiation decreased in all three cases. This effect was most pronounced in osteogenic differentiation; however, till passage 5 the differentiation still occurred on satisfactory level. In case of adipogenic differentiation some cells even from passage 10 differentiated, but the efficiency of this process dropped clearly after passage 6.
Capillary-like structures were formed by cells up to passage 8 (with decreasing efficiency), but the ones formed by cells older than passage 5 disrupted after 24 hours (in contrast to cells from earlier passages). Furthermore, cells older than passage 6 did not survive the Matrigel culture.
In this study we showed that in similar way to osteogenic and adipogenic differentiation, differentiation of hMSCs towards endothelial lineages declines during culture expansion.

MONDAY
Genetic modification of hematopoietic cells – challenges and prospects
Christopher Baum , Axel Schambach, Ute Modlich, Bernhard Schiedlmeier, Melanie
Galla, Tobias Mätzig, Martijn Brugman, Stefan Bartels, Olga Kustikova, Christine
Voelkel, Julia Suerth, Johannes Kühle, Dietrich Lesinski
Adverse events related to insertional mutagenesis may compromise the therapeutic prospects of gene therapy in the hematopoietic system. Major progress in the understanding of the underlying mechanisms has opened rational approaches to increase the therapeutic index with improved vector technology. We have established sensitive nonclinical models, in transplanted C57BL/6J mice or using cultured cells, to reveal the transforming capacity of insertional activation of cellular proto-oncogenes such as Evi1 or
Prdm16 , and developed integrating gene vectors designed to reduce the risk of insertional transformation by altering the insertion pattern and the expression cassette of the transgene. Our experiments identified a hierarchy of factors that determine the emergence of insertional mutants. In transplanted mice, the progeny of hematopoietic cells with sustained self-renewal potential is considerably more sensitive to insertional mutations than multipotent progenitor cells and more mature myeloid cells. The second most important factor in the induction of insertional mutants is the presence of a strong enhancer-promoter in the integrated transgene, which may act over distances exceeding
100 kb to activate neighbouring proto-oncogenes. The third factor is the insertion pattern of retroviral vectors: gammaretroviral vectors with their preferred insertion in regulatory gene regions induce a greater risk than lentiviral vectors with their preferences for actively transcribed gene regions. However, lentiviral vectors are still able to trigger insertional transformation, indicating the need to develop vectors with an even more neutral or potential site-specific integration mechanism. To this end, we and others embarked on alpharetroviral vectors or hybrid systems relying on transposases.
In recent studies, we investigated the fate of insertional mutants if cells were cultured using a novel cytokine cocktail developed by Zhang and Lodish to support self-renewal of hematopoietic stem cells. High-throughput integration site analysis performed at consecutive time points showed polyclonal reconstitution of hematopoiesis (several thousands insertion sites identified in an experiment involving 56 C57BL/6J mice) without overt clonal skewing triggered by potentially transforming lentiviral insertions in cellular proto-oncogenes. We hypothesize that the establishment of polyclonal hematopoiesis, supported by the use of improved cytokine conditions in gene transfer protocols, may repress insertional mutants.
Finally, hematopoiesis derived from induced pluripotent cells emerges as a novel avenue to treat diseases by gene-modified cells. We have developed a genetically reversible retrovirus-based system that induces pluripotency at high efficiency. A major challenge remains their differentiation to hematopoietic cells with robust engraftment potential.

MONDAY
Gene therapy for inherited immunodeficiency: successes and future challenges
Adrian Thrasher
Somatic gene therapy has been proposed as an exciting new therapeutic strategy for treatment of human genetic disease. Four recent studies have demonstrated highly effective gene therapy for the X-linked form of SCID (SCID-X1) and ADA-deficiency, using retroviruses to deliver the therapeutic genes into haematopoietic stem cells ex vivo.
X-linked severe combined immunodeficiency (X-SCID), caused by mutations in the common cytokine receptor gamma chain, is invariably fatal in the absence of therapeutic bone marrow transplantation. In the presence of HLA-matched family donors, the majority of patients can expect to be cured. For other donor sources, particularly parental, success rates are reduced, and full restoration of immunity is often not achieved. We have treated ten children with SCID-X1 by gene transfer to bone marrow stem and progenitor cells using a gibbon ape leukaemia virus (GALV)-pseudotyped retroviral vector. All patients have developed substantial immunity, with a normally complex T cell spectratype determined by CDR3 PCR, although in one patient recovery has been partial.
Clinical response has been excellent and all are leading normal lives at home. Similar responses have been observed in patients with ADA-deficiency. However one patient with SCID-X1 has also developed lymphoproliferative malignancy as a result of insertional mutagenesis, highlighting the need to develop improved assay systems to assess risk, and for enhanced vector design. These refinements have led to the design of new clinical trials, and to the investigation of gene transfer vectors with novel configurations.

TUESDAY
Negative feedback signaling in intestinal epithelial homeostasis
Gijs R. van den Brink
Differentiated cells in rapidly renewing tissues such as epithelia of the gastrointestinal tract are in a dynamic equilibrium with precursor cells in order to balance the rate of proliferation with cell loss at the epithelial surface. The balance between input and output in homeostatic dynamic equilibria depends on the presence of negative feedback loops. In recent years we have learned much about the way Wnt signaling specifies the fate and proliferation of intestinal epithelial precursor cells. Much less is known about the mechanisms in place to control Wnt signaling.
We found that Indian Hedgehog (Ihh) secreted by differentiated epithelial cells at the luminal surface of the intestine. Conditional activation of Hedgehog signaling results in loss of Wnt signaling with depletion of intestinal precursor cells which undergo premature differentiation to the enterocyte lineage. Conversely, conditional loss of Ihh resulted in increased Wnt signaling and accumulation of precursor cells with lengthening and multiplication of the intestinal crypts. Loss of Ihh not only resulted in epithelial changes that are characteristic of an intestinal wound healing response but additionally in the recruitment of macrophages and fibroblasts two other typical features of wound healing. Prolonged loss of Ihh resulted in progressive leukocyte infiltration of the crypt area, the development of mucosal damage and intestinal fibrosis.
Our data show that Ihh is a negative feedback regulator of intestinal precursor cell fate and that its loss results in the activation of multiple aspects of a wound healing response which ultimately results in the development of chronic inflammation and fibrosis. Thus
Ihh is a signal derived from the superficial epithelial cells that may act as a critical indicator of epithelial integrity.

TUESDAY
The competitive nature of stem cells
Eduardo Moreno
Cell competition is a short-range cell-cell interaction leading to the proliferation of winner cells at the expense of losers, although either cell type shows normal growth in homotypic environments. Drosophila Myc is a potent inducer of cell competition. I will present data showing that stem cells with relative lower levels of dMyc are replaced by
GSCs with higher levels of dMyc. By contrast, dMyc-overexpressing GSCs outcompete wild-type stem cells without affecting total stem cell numbers. A naturally occurring cell competition border formed by high dMyc-expressing stem cells and low dMyc-expressing progeny. We propose that dMyc-induced competition plays a dual role in regulating optimal stem cell pools and sharp differentiation boundaries, but is potentially harmful in the case of emerging dmyc duplications that facilitate niche occupancy by pre-cancerous stem cells. Moreover, competitive interactions among stem cells may be relevant for the successful application of stem cell therapies in humans.

TUESDAY
Colon cancer stem cells: isolation & identification
Kristel Kemper , Louis Vermeulen, Felipe De Sousa e Melo, Dick Richel, Giorgio
Stassi, Jan Paul Medema
The classical tumour model, suggesting that each tumour cell is capable of initiating a new tumour, is nowadays replaced by the cancer stem cell principle, suggesting that only a subset of tumour cells are responsible for tumour growth. These tumour initiating cells, also called cancer stem cells (CSCs) have been studied widely in the last couple of years.
A CSC is defined as a cell that can form tumours in vivo which can be propagated several passages as well as a cell that can differentiate into multiple lineages.
In colon cancer, CSCs have been identified by expression of markers like CD24, CD44,
CD166 and CD133. This last marker, CD133, has been under a lot of debate, because its expression was found both in CSCs as well as differentiated cells. Our group showed that
CD133 is expressed on differentiated cancer cells, but the protein is probably differentially folded on these cells compared to CSCs and therefore the epitope used to identify CSCs, is masked in this setting. Unmasking can be done by different treatment of tissue which explains the broad staining of CD133 on tissue sections. CD133 can thus be used as a CSC marker but should be used with caution.
The function of most markers used for CSC isolation, like CD133, is still unknown. To identify a more functional CSC marker, our group used the observation that, even though colorectal cancer have mutations in APC or B-catenin that should constitutively activate the Wnt signalling cascade, most colorectal cancers still display cellular heterogeneity in nuclear localization of B-catenin, indicating that the Wnt regulating is more complex.
Addressing this regulation, a Wnt reporter construct was used to identify a population that had high Wnt activity and displayed CSC features. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate β-catenin-dependent transcription and subsequently
CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the
CSC phenotype in more differentiated tumour cells both in vitro and in vivo. Altogether, these data show that high Wnt activity can be used as a more functional CSC marker and that activity of Wnt and thus the stemness of colon cancer cells is in part orchestrated by the microenvironment. This regulation of stemness is therefore much more dynamic than previously expected.

TUESDAY
Nestin + MSCs: self-renewing stem cells with HSC niche functions
Simón Méndez-Ferrer
, Tatyana V. Michurina, Francesca Ferraro, Amin R. Mazloom,
Ben D. MacArthur, Sergio A. Lira, David T. Scadden, Avi Ma’ayan, Grigori N.
Enikolopov and Paul S. Frenette
The identity of the cells forming the haematopoietic stem cell (HSC) niche remains unclear. Bone-lining osteoblasts and preosteoblasts, osteoclasts, megakaryocytes, endothelial and reticular cells, as well as mesenchymal progenitors have been proposed to contribute to the HSC niche. However, despite their broad therapeutic potential, mesenchymal stem cells (MSCs) and their putative functions in the HSC niche remain poorly defined due to MSCs heterogeneity, the inability to assess their in vivo selfrenewal and the paucity of specific markers that allow for their identification, isolation and genetic manipulation. Previous work from the Frenette lab has shown that HSC mobilization induced by granulocyte colony-stimulating factor (G-CSF) requires signals from the sympathetic nervous system and is associated with inhibition of bone-lining osteoblasts. However, physiological release of HSCs into the bloodstream follows circadian oscillations of Cxcl12/Sdf-1 expression triggered by cyclical activation of the
β
3
-adrenergic receptor, which is not expressed by osteoblasts but other stromal elements
(Mendez-Ferrer et al . 2008; Nature 452:442-7). We have recently identified these stromal cells as bona fide MSCs identified using regulatory elements of the intermediate filament protein nestin. In the bone marrow of Nestin Gfp transgenic mice, CD31 CD45 GFP + peri-vascular cells expressing nestin are anatomically associated with HSCs and sympathetic fibres. They are highly enriched in the expression of HSC maintenance genes, which are selectively downregulated in this cell population following G-CSF treatment or
β
3
-adrenergic receptor stimulation. Although they represent a small subset of stromal cells, Nestin:GFP
+
cells contain all the colony-forming unit-fibroblastic (CFU-F) activity and have the exclusive capacity within bone marrow cells of forming multipotent clonal spheres able to self-renew, differentiate into mesenchymal lineages and transfer haematopoietic activity in serial transplantations using heterotypic bone ossicles. The proliferation and osteoblastic differentiation of Nestin:GFP
+
cells is regulated by cytokines (G-CSF), neural (sympathetic) and hormonal (PTH) input. Lineage-tracing studies in Nes-Cre / R26R mice have shown the contribution of nestin
+
cells in endochondral and membranous ossification. Administration of tamoxifen to adult Nes-
Cre
ERT2
mice bred to a reporter line has revealed the presence of GFP
+
osteolineage cells after 8-month chasing, suggesting an active role for adult nestin
+
MSCs in physiological bone turnover. In vivo nestin
+
cell depletion using Nes-Cre
ERT2
mice bred to a Creinducible diphtheria toxin receptor line rapidly reduces HSC content in the bone marrow, owing at least partially to their mobilization to extramedullary sites. Purified HSCs injected into lethally irradiated mice rapidly home near bone marrow nestin
+
MSCs, whereas in vivo nestin
+
cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results indicate that nestin regulatory elements may represent a valuable tool to prospectively isolate and genetically manipulate MSCs. They also uncover a close relationship between two different somatic stem cells and reveal essential functions for MSCs in the HSC niche.

TUESDAY
Dynamic Interactions Between the Nervous and Immune Systems with the
Microenvironment, Regulate Normal and Leukemic Human Stem Cells
Tsvee Lapidot
Functional, preclinical models for normal and leukemic human stem cells using immune deficient NOD/SCID mice as transplantation recipients revealed that homing and repopulation are dependent on SDF-1/CXCR4 interactions. These in vivo models can predict clinical outcome since SDF-1 induced migration potential of human CD34+ progenitors correlates with their repopulation in autologous transplanted patients and surface CXCR4 expression levels are a poor prognosis factor for leukemic patients. Stem cell homing, retention, release and mobilization are tightly regulated processes, involving bone turnover and an interplay between cytokines, chemokines, adhesion molecules and proteolytic enzymes. The roles of CD44, MT1-MMP and RECK in human stem cell migration, homing, retention and G-CSF induced mobilization will be discussed.
Most blood forming stem cells are retained in the bone marrow (BM), anchored to specialized niches via adhesion interactions, which prevent their motility and proliferation. However, low levels of motile progenitors migrate in the circulation as part of homeostasis. These low levels are dramatically amplified during alarm situations in response to stress signals due to injury, bleeding and infections, as part of host defense and repair mechanisms. These stress signals are mimicked by repeated G-CSF stimulation in order to mobilize stem and progenitor cells to the circulation in order to harvest them for clinical transplantation protocols. The roles of HGF and c-Met in ROS production during G-CSF induced mobilization will be discussed. Stem cell adhesion interactions with the stromal niche supporting cells need to be dynamic in order to allow the undifferentiated cells to proliferate, differentiate and migrate. Osteoclasts have a dual role in host defense: bone remodeling and regulation of stem cells by freeing them form their inhibitory anchorage to stromal cells. Thus, osteoclast/osteoblast interactions also regulate
BM leukocyte production on demand. Osteoclasts, osteoblasts, as well as stem and progenitor cells functionally express receptors for neurotransmitters. Immature human
CD34+ cells dynamically express dopamine and epinephrine receptors and inflammatory, myeloid cytokines such as G-CSF and GM-CSF increase catecholaminergic receptor expression in order to facilitate leukocyte production and trafficking. This up regulation activates the progenitor cells in response to stimulation by the neurotransmitters and induces their motility and proliferation via Wnt signaling. Thus , regulation of leukocyte production and trafficking by stem cells in the BM reservoir is dynamic and involves mutual, reciprocal interactions between the nervous and immune systems with the stromal microenvironment throughout the body. Stimulations with the cytokine bFGF induce proliferation and self renewal of murine stem cells via the JAK/STAT pathway and also promote proliferation of human myeloid leukemia cells and inhibition of their differentiation via ROS inhibition. Finally, while various types of bone marrow stromal cells including osteoblasts, endothelial, reticular and nestin+ cells were identified as stem cell niche supporting cells, in addition to osteoclasts, we will discuss preliminary results on bone marrow Mac-1+/CD45+ dendritic cells which regulate hematopoietic stem cells.

Model Systems to Elucidate Molecular Mechanisms in Stem Cell Niches
TUESDAY
Kateri Moore
How stem cells maintain normal tissue homeostasis and respond to systemic physiological stimuli are questions of central importance in stem cell biology. All stem cells have the ability to self-renew and to differentiate to produce functional mature cells. These decisions have to be balanced and carefully regulated in order to ensure a life-long source and supply of differentiated cells. Stem cell microenvironments or niches play important, though poorly understood, roles in regulating the properties of stem cells. We suggest that hematopoietic stem cells (HSC) are maintained in a state of relative quiescence within a specific cellular architecture and are infrequently utilized during steady-state hematopoietic homeostasis. Within the niche there is a unique molecular dialog that normally maintains quiescence but can mediate rapid HSC activation and cell fate decisions in response to systemic stimuli. We propose that niche mediated quiescence is central to the maintenance of HSC with robust self-renewal capability and that this property is progressively depleted as a function of proliferative history. We have addressed these questions by utilizing mouse genetic models. One model allows stem cell specific and controllable dynamic chromosomal labeling with GFP through which viable label-retaining cells (LRC) can be isolated for functional and molecular studies. We have developed a molecular profile of the LRC on the basis of their divisional history and also show that the ability of LRC to repopulate decreases as cells divide and dilute the GFP-label irrespective of canonical stem cell phenotypic markers. Another transgenic mouse system perturbs signaling from niche specific cells and has detrimental effects on the maintenance of the resident stem cells after systemic insult. These mice constitutively over-express a Wnt-inhibitor, Wif1, specifically in osteoblasts. Using this model we have shown that stem cells appear to proliferate at the expense of self-renewal via a dysregulation of niche signaling impacting on unexpected pathways. Ultimately, we hope to develop a precise portrait of the molecular and cellular interactions characteristic to HSC and niche cells. This information will inform strategies that target the niche as a therapeutic entity.

TUESDAY
Substrate Elasticity Regulates Skeletal Muscle Stem Cell Self Renewal in Culture
Stéphane Corbel,
P. Gilbert, K. Havenstrite, A. Sacco, K. Magnusson, N. Leonardi,
P. Kraft, N. Nguyen, S. Thrun, M. Lutolf, H.M. Blau.
Procedures for isolating enriched populations of adult muscle stem cells (MuSCs) were recently developed by our laboratory and others. Transplantation studies revealed an extraordinary potential of MuSCs to contribute to muscle fibers, and to access and replenish the satellite cell compartment. However, MuSCs are a relatively rare cell type and their stem cell properties are rapidly lost once plated in culture limiting their clinical utility. We hypothesized that the long term culture of MuSCs on surfaces mimicking key features of the in vivo microenvironment would be able to increase their viability, promote division, and maintain their in vivo function. To this end, we employed a novel bioengineered hydrogel culture platform in conjunction with a newly developed automated image analysis computer algorithm and assayed the in vitro and in vivo behavior of MuSCs cultured on hydrogels with different biophysical and biochemical microenvironmental cues. We found that culture upon compliant hydrogel that mimicks muscle elasticity greatly enhances MuSC viability and prevents their differentiation compared to rigid plastic. Further, transplantation studies revealed that MuSCs exposed to a compliant hydrogel surface with mechanical properties similar to native skeletal muscle self renew and retain similar regenerative potential to freshly isolated MuSCs. Our results establish parameters for the long term culture and self renewal of MuSCs in vitro that maintain MuSC function in vivo , which is essential to translation of MuSC biology to the clinic for treatment of human muscle degenerative disorders, and present a culture paradigm for other adult tissue specific stem cell types.

TUESDAY
Pursuing Pluripotency
Ihor R. Lemischka
Embryonic stem (ES) cells represent an attractive model system to elucidate the molecular and cellular mechanisms responsible for cell fate decisions. These cells also hold great promise for the future of regenerative medicine. Much progress has been made in recent years to elucidate the mechanisms that control ES cell pluripotency, as well as lineage-specific commitment. Much remains to be elucidated, and there is currently no systems level “picture’ of how cell fate decisions occur in response to defined input signal, and as a function of time. We have embarked on efforts to first: identify most, if not all, regulatory components that mediate cell fate decisions in murine ES cells, and second: to develop methodologies to analyze a cell fate decision as it occurs over time following a defined stimulus, and at multiple biochemical/molecular levels. The experimental platform that underlies most of our studies is controlled, short hairpin
(sh)RNA-mediated down regulation of important cell fate regulators. We have analyzed a cell fate decision process at the levels of changes in chromatin architecture, transcriptional activity, steady-state mRNA populations, and the entire nuclear protein population. In addition, we have performed these analyses as a function of time.
Numerous interesting observations have been obtained, and a synthesis of our numerous observations represents a first systems level “picture” of a change in stem cell fate. We have also embarked on similar studies in the human ES cell system, as well as in ES-like cells derived from human fibroblasts using recently reported reprogramming approaches.

WEDNESDAY
Generation of human iPS cells from cord blood, long term expansion in suspension culture and differentiation into functional cardiomyocytes
Ulrich Martin
Induced pluripotent stem (iPS) cells may represent a favoured cell source for future regenerative therapies. We have generated human iPS cells from cord blood (CB). CBiPS cells show typical characteristics of embryonic stem cells and can be differentiated into derivatives of all three germ layers both in vitro and in vivo . For future therapeutic production of patient-specific and allogeneic iPS cell derivatives, CB may provide a juvenescent cell source with biological superiority, which is harvested routinely and without donor risk for public and commercial CB banks.
However, therapeutic application of iPS derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have therefore established the expansion of undifferentiated human embryonic stem
(hES) and hiPS cells in suspension culture using a defined culture medium. HES / hiPS cells were expanded for up to 20 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers including functional cardiomyocytes. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.

WEDNESDAY
Programming and Reprogramming: New Approaches for Understanding Disease
Chad Cowan
Our research is focused on understanding the contribution of environmental and genetic factors in the development of obesity and obesity related diseases. We seek to gain a better understanding of these diseases by capturing the disease genotype through the creation of induced pluripotent patient-specific stem cells. These cells can then be used to interrogate the disease phenotype in differentiated adult cells types associated with or affected by disease. By comparing cells from healthy donors and obese donors we hope to discover the cellular origins of obesity and use this information to aid in the battle against this and related metabolic diseases. Our progress towards this goal will be the topic of my presentation.

WEDNESDAY
In vitro Acquisition of Pluripotency in Primordial Germ Cells
Toshio Suda, Go Nagamatsu
Induced pluripotent stem (iPS) cell technology allows somatic cells to acquire pluripotency. However, a low efficiency of conversion, coupled with a long culture period for iPS cell formation, has made it difficult to investigate the mechanisms involved in the reprogramming of differentiated cells. To understand the mechanisms underlying the acquisition of pluripotency, this study focused on unipotent primordial germ cells
(PGCs), which can be reprogrammed to pluripotent embryonic germ (EG) cells at high efficiency and faster than other cells under defined conditions. The use of small molecule combinations, including the ERK inhibitor, GSK3-β inhibitor, and TGF-βtype-1 receptor inhibitor, induced PGCs to enter the pluripotent state at an efficiency of approximately
12.1% by day 10 of culture. Fluorescent activated cell sorting (FACS) identified the progress of the cell populations towards the pluripotent state, and facilitated the analysis of gene expression changes in pluripotent candidate cells during the acquisition of pluripotency. In addition to the loss of characteristics associated with germ cell specificity, the expression levels of pluripotent cell-associated genes changed on day three following the induction of pluripotency. The data suggested that, during the putative induction phase, a specific ratio of three reprogramming factors (Oct3/4, Sox2 and Klf4) is required for the acquisition of pluripotency, not only in the development of PGCs to
EG cells, but also in somatic cell reprogramming. In addition, two additional reprogramming factors, Prmt5 and Tbx3, were identified during the putative induction phase. Prmt5 could replace Sox2, while Tbx3 resulted in alkaline phosphatase (ALP) positive ES cell-like colonies when used together with Klf4, Sox2 and c-Myc.

WEDNESDAY
Live imaging of the first hematopoietic stem/progenitor cells emerging in the mouse embryonic aorta.
Jean-Charles Boisset, Gert van Cappelen, Charlotte Andrieu, Niels Galjart,
Elaine Dzierzak, Catherine Robin
During mouse development, hematopoietic stem cells (HSC) are first generated in the dorsal aorta at mid-gestation (E10.5). HSCs are found one day later in other highly vascularized organs: the yolk sac, placenta and fetal liver. Clusters of cells attached to the wall of the aorta are found in many species. Because such clusters are totally absent in hematopoietic mutants, devoid of HSCs, it is believed that these clusters contain hematopoietic progenitors and HSCs.
The origin of HSCs has long been controversial but studies performed in the chicken and more recently in mouse models highlight a specialized endothelial cell population, the hemogenic endothelium, as the direct HSC precursor. However, visualization of the dynamic events during which hemogenic endothelial cells transit to hematopoietic cells have never been observed in vivo because of the deep localization of the aorta in the opaque mouse embryo. By combining a new dissection procedure to access the aorta with time-lapse confocal imaging, we show the first live and dynamic imaging of the endothelial to hematopoietic cell transition in the physiological context of the mouse embryonic aorta. We show that the newly generated cells, budding directly from the ventral endothelium to the aortic lumen, emerge at the time of HSC activity (E10.5) and express HSC markers (Sca1
+
, c-kit
+
, CD41
+
). Thus we visualize for the first time in the mouse embryo the emergence of the first hematopoietic stem/progenitor cells from the hemogenic endothelium.

WEDNESDAY
Diverse stem cell projects supported by the nystem shared facility grant at MSSM
Liang Dong, Vera Alexeeva, Ihor Lemischka, Sunita DSouza
The advent of induced pluripotent stem cell (iPSC) technology will usher in an era of cell based therapeutic approaches. The success of cell-based therapies is based on the ability to generate a continuous supply of large numbers of patient-specific differentiated cell types. A necessary precursor towards this goal is the efficient generation of insertion-free patient-specific iPSCs as well as systematic studies of genes/growth factors necessary to promote differentiation into various tissue specific progenitors. With this objective in mind, the NYSTEM-funded, human embryonic stem cell (hESC)/induced pluripotent stem cell (iPSC) Shared Resource Facility (SRF) was established in 2008 at Mount Sinai
School of Medicine (MSSM). Such a facility is becoming increasingly essential as many physician-scientists are drawn to ESC/iPSC technology to study the etiology of the development of their favorite disease. To facilitate the transfer of this technology to these labs, hESC/iPSC SRF regularly conducts classes to teach physician-scientists how to generate iPSCs and the methods involved in maintaining and differentiating ESCs/iPSCs into the lineage of choice. Scientists are also provided with tested stem cell reagents at vastly discounted pricing made possible due to the establishment of two stem cell supply centers, bulk purchasing and NYSTEM funding.
In addition, since not all labs have the necessary funds to establish ESC technology, the hESC/iPSC SRF assists these labs by providing them with the cell types necessary for their studies. For e.g. cardiomyocytes are provided to aid in signaling studies and hepatocytes are regularly supplied to aid in the development of an in vitro hepatitis C virus (HCV) model of infection. To further the development of iPSC technology, the hESC SRF has constructed a humanized version of the murine piggyBac vector system in collaboration with Dr. Christoph Schaniel from Dr. Lemischka’s lab. This technology will aid in the creation of patient-specific iPSCs lines that are completely free of exogenous
DNA. Realizing the full potential of hESC/iPSC technology mandates the development of an efficient means to generate numerous reporter cell lines as well as inducible over expression systems. We are currently exploring zinc finger technology as a means towards this end. The hESC/iPSC SRF is also actively collaborating with the Memorial
Sloan Kettering Cancer Center (MSKCC) Core Facility in New York to improve current neuronal development protocols. In exchange the hESC/iPSC SRF provides them with necessary know-how in endoderm/cardiac differentiation studies. These collaborations are not restricted to academia but also extend to industry. In collaboration with various companies, the hESC/iPSC SRF is currently involved in the construction of various reporter cell lines for Drs. Ihor Lemischka, Hans Snoeck, Valerie Evans and Dr. Woo. In conclusion, the NYSTEM-funded hESC/iPSC SRF has been established and provides valuable service to the MSSM community by providing scientists with direct access to the latest technology developments in the dynamic field of stem cell research.

WEDNESDAY
Non-coding RNAs: guardians of development?
Selina van Leeuwen, Larysa Pevny, Jonas Frisén,
Harald Mikkers
Long non-coding RNAs have been food for thought for a long time. Initially the majority was regarded as “transcriptional noise” but recently more and more evidence has emerged that evolutionary conserved long non-coding RNAs are important transcriptional regulators. We have identified two conserved long non-coding RNAs, sharing multiple exons, in the Sox2 locus upstream of Sox2 . The transcription factor SOX2 is a major player guiding and instructing the self-renewal machinery of embryonic and neural stem cells, and preventing differentiation. We have started to characterize these non-coding transcripts that are located in the Sox2 locus using different approaches. Initial experiments revealed an expression pattern that is restricted to neural tissues and have pointed to a direct correlation between Sox2 transcription and the transcription of these non-coding RNAs. These observations have suggested a direct regulation of Sox2 transcription by upstream located non-coding RNAs. To investigate this we altered the endogenous loci of the non-coding RNAs in embryonic stem cell lines by homologous recombination. Here the results obtained from the different experiments set out to determine the role of these non-coding RNAs located in the Sox2 locus will be discussed.

WEDNESDAY
Efficient ZFN-based gene inactivation in transgenic human iPS cells as a model for gene editing in patient-specific cells
Sylvia Merkert , Kafaitullah Khan, Alexandra Haase, Kristin Schwanke, Toni Cathomen,
Ulrich Martin
Gene targeting by homologous recombination via customized zinc-finger nucleases
(ZFN) is a powerful method to manipulate the genome and correct genetic defects.
Although efficiency of ZFN based homologous recombination has been shown to be significantly higher than by means of conventional gene targeting, the selection of suitable clones still requires cells that proliferate in culture. Clinically applicable ZFNbased gene correction in patient-specific cells was hardly possible so far, due to the inability to sufficiently expand most adult (stem and progenitor) cells in vitro . However, the availability of human induced pluripotent stem (hiPS) cells with their almost unlimited potential for proliferation and differentiation now offers novel opportunities for the development of patient-specific regenerative therapies. As a first step towards ZFNbased gene targeting, a non-viral gene-transfer approach with transfection rates of up to
60% and high cell vitality was established for hiPS cells. Aiming at the development of a general ZFN-based recombination approach in hiPS cells, we investigated the functionality of an eGFP specific ZFN in an eGFP transgenic hiPS cell clone. Targeting of the eGFP via non-homologous end joining resulted in up to 3% eGFP neg
cells, and mRNA expression of genetically modified eGFP was shown in sorted eGFP neg
hiPS cell clones.
Ultimately, the development of a generally applicable protocol for ZFN based sitespecific recombination and gene correction in patient-specific hiPS cells may enable the development of cellular therapies for various genetic diseases.

WEDNESDAY
Induced Pluripotent Stem Cells as Models of Diseases
Christoph Schaniel, Xonia Carvajal-Vergara, Sherly Pardo, Sonia Mulero-Navarro,
Sunita D’Souza, Bruce Gelb, Eric Adler, Ihor R. Lemischka
The generation of induced pluripotent stem cells (iPSCs) from somatic cells of patients with defined genetic disorders holds considerable hope for establishing surrogate models of human diseases. Pluripotent stem cells, including human embryonic stem cells (hESCs) and hiPSCs, have the ability to self-renew and differentiate into all cell types of the body.
Therefore, pluripotent stem cells can provide us with an unlimited source of biomaterial, and represent an ideal research tool to understand the etiology of complex diseases, for drug discovery and toxicology screening, and ultimately for cell-based therapies.
We have established and characterized hiPSC lines from patients with Noonan syndrome
(NS) and related disorders, including LEOPARD syndrome (LS). These syndromes are inherited autosomal dominant developmental disorders affecting multiple organs, including the heart, and are caused by single gene mutations in members of the RAS-
MAPK signaling pathway. In the case of LS, patient-specific iPSC-derived cardiomyocytes show hypertrophic features in vitro , one of the characteristics of this disorder. NS iPSC-derived cell types are currently being investigated for patient-specific phenotypes. We are also interested in long QT syndromes (LQTS). LQTS are associated with delayed repolarization of ventricular cells in the heart detected as abnormally long
QT intervals on an electrocardiogram. Currently, LQTS are associated with mutations in
12 different genes. In the case of LQT2, which accounts for 35-45% of all LQTS, the affected gene is KCNH2 , which encodes for the potassium voltage-gated channel, hERG.
We have already generated and characterized several iPSC lines from a patient with
LQT2 syndrome caused by a splice site mutation in KCNH2 . The iPSC lines have been differentiated into cardiomyocytes and are being analyzed for their functionality using calcium imaging and patch clamp recordings. These data should reveal the consequences of the mutation on cardiomyocyte function, including a LQT2 phenotype. These studies demonstrate the usefulness of the iPSC technology for disease modeling with the potential for development of novel tools for the diagnosis and treatment of cardiac diseases and disorders.

WEDNESDAY
Derivation of induced pluripotent (iPS) cell lines from patients with cardiovascular disease
Christian Freund , Richard Davis, Daniela Salvatori, Saskia Maas, Dorien Ward-van
Oostwaard, Roy Nauw, Kees Westermann, Frans Disch, Danielle de Jong, Karoly Szuhai,
Hans Tanke, Christine Mummery
Recently it has been shown that human somatic cells can be reprogrammed to an embryonic stem cell(ESC)-like state by overexpression of four transcription factors
(induced pluripotent stem cells, iPS cells). Like human embryonic stem cells, iPS cells are pluripotent and can differentiate into derivatives of the three germ layers in vitro and in vivo. With the development of the iPS cell technique it has become possible to generate pluripotent stem cells from patients with genetic diseases. We are interested in vascular diseases, specifically in hereditary hemorrhagic telangiectasia (HHT), where patients have weak blood vessels caused by defective transforming growth factor (TGF)-β signaling in endothelial cells.
Here we describe the generation of four iPS cell lines from skin fibroblasts of two healthy human individuals and two HHT-patients by retroviral overexpression of the four transcription factors OCT-3/4, SOX-2, KLF-4 and C-MYC. Human iPS cell lines could be maintained under feeder-free conditions on matrigel in the presence of mTESR1 medium for at least 13 passages without accumulation of karyotypic abnormalities as determined by COBRA assay. iPS cells showed a homogenous expression of typical hESC markers, e.g. OCT-3/4, SOX-2, NANOG, TRA-1-81, SSEA3 and SSEA4 as assessed by immunostaining. The expression of additional stem cell markers was confirmed by semi-quantitative RT-PCR.
When the iPS cell lines were cultured as embryoid bodies (EB’s) they gave rise to derivatives of the three germ layers as determined by immunostaining (ectoderm: βIII-
TUBULIN, GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP), endoderm: α-
FETOPROTEIN (AFP), mesoderm: VIMENTIN, α-SMOOTH MUSCLE ACTIN
(SMA)). Pluripotency of iPS cells in vivo was confirmed by teratoma formation in SCID mice.
For the directed differentiation of iPS cells into the vascular lineage, EB’s were cultured in the presence of 20% serum and 50ng/ml VEGF for 14 days. The presence of endothelial cells and smooth muscle cells was confirmed by immunostaining for PECAM,
VE-CADHERIN, ENDOGLIN, VON WILLEBRAND FACTOR or α-SMA, respectively. Currently we are investigating whether derivatives of patient iPS cells reflect features of the disease in vivo.

in vivo.
WEDNESDAY
Mouse Pluripotent Stem Cell Models of a human cardiac sodium channelopathy
Richard Davis , Cathelijne van den Berg, Simona Casini, Christian Freund,
Leon Tertoolen, Carol Ann Remme, Connie R. Bezzina, Christine Mummery
Mutations in the gene encoding the cardiac sodium channel, SCN5A , have been implicated in multiple cardiac arrhythmia syndromes, such as long-QT syndrome type 3
(LQT3) and Brugada syndrome. Several transgenic mouse models carrying the mouse equivalent of human SCN5A mutations have been generated, which recapitulate the disease phenotype observed in humans. With the generation of induced pluripotent stem
(iPS) cells by the reprogramming of somatic cells into an embryonic stem cell (ESC)-like state, it may now be possible to generate these disease models directly from individuals carrying the mutation. However, it remains unclear whether iPS cell derivatives can indeed recapitulate the phenotype of cardiac diseases in vitro. This study aims to compare cardiomyocytes derived from both mouse ESCs and iPS cells that carry the mouse equivalent (1798insD) of the human SCN5A-1795insD mutation ( Scn5a
1798insD/+
), and to determine whether they display a similar electrophysiological phenotype to that of the transgenic mouse model. Mouse iPS (miPS) cells were generated by retroviral overexpression of the four transcription factors Oct3/4, Sox2, Klf4 and c-Myc. miPS cells resembled mESCs in morphology and proliferation rate, and expressed pluripotency markers as assessed by immunostaining. Scn5a +/+ and Scn5a 1798insD/+ mESCs and miPS cells were differentiated into cardiomyocytes by the formation of embryoid bodies.
Spontaneously contracting cells were identified in culture that morphologically resemble cardiomyocytes. Electrophysiological analysis of the mESC lines revealed that cardiomyocytes derived from Scn5a
1798insD/+
display a reduced sodium current density compared to wildtype mESC-derived cardiomyocytes, recapitulating observations made

PARTICIPANTS
Dr. Nick Barker
Hubrecht Institute
Uppsalalaan 8
3584 CT Utrecht
The Netherlands n.barker@hubrecht.eu
Stephane Corbel PhD
Stanford University
Baxter Laboratories for Stem Cell Biology
Stanford 94305
USA scorbel@stanford.edu
Cheryl Dambrot MSc
LUMC
Dept. of Cardiology
Albinusdreef 2, C5-P
2333 ZA Leiden
The Netherlands
C.Dambrot@lumc.nl
Prof. Elaine Dzierzak
Erasmus Stem Cell Institute
Dept. Of Cell Biology
P.O. Box 2040
3000 CA Rotterdam
The Netherlands e.dzierzak@erasmusmc.nl
Prof. Salima Hacein-Bey-Abina
Hopital Necker
Dept. of Biotherapy
149 rue de Sevres.
75743 Paris Cedex 15
France salima.hacein-bey@nck.aphp.fr
Prof. Stefan Karlsson
Lund University
Molecular Medicine
BMC A12
Lund 221 84
Sweden
Stefan.Karlsson@med.lu.se
Prof. Christopher Baum
Medical School Hannover
Dept. of Experimental Hematology
Carl-Neuberg Strasse 1
30625 Hannover, Germany
Baum.Christopher@mh-hannover.de
Dr. Chad Cowan
Massachusetts General Hospital
Simches Research Center
185 Cambridge Street
Boston, MA 02114
USA ccowan1@partners.org
Dr. Richard Davis
LUMC
Dept. of Anatomy & Embryology
Albinusdreef 2, S1-P
2333 ZA Leiden
The Netherlands
R.P.Davis@lumc.nl
Dr. Christian Freund
LUMC
Dept. of Anatomy & Embryology
Albinusdreef 2, S1-P
2333 ZA Leiden
The Netherlands
C.M.A.H.Freund@lumc.nl
Karolina Janeczek Portalska MSc
University of Twente
Institute for Biomedical Technology
Drienerlolaan 5
7522 NB Enschede
K.K.Janeczek@utwente.nl
Kristel Kemper MSc
AMC
Meibergdreef 9
1105 AZ Amsterdam
The Netherlands k.kemper@amc.uva.nl

Prof. Frits Koning
LUMC
Dept. of Immunohematology and
Blood Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
F.Koning@lumc.nl
Prof. Ihor Lemischka
Mount Sinai School of Medicine,
One Gustave Levy Place, Box 1496
New York, New York 10029-6574
USA ihor.lemischka@mssm.edu
Simón Méndez-Ferrer PhD
Mount Sinai School of Medicine
One Gustave L Levy Place
New York, NY 10029
USA simon.mendez-ferrer@mssm.edu
Kenichi Miharada PhD
Lund University
Molecular Medicine and Gene Therapy
BMC A12
Lund 221 84
Sweden
Kenichi.Miharada@med.lu.se
Dr. Kateri Moore
Mount Sinai School of Medicine
One Gustave Levy Place, Box 1496
New York, New York 10029-6574
USA kateri.moore@mssm.edu
Ir. Marinus Nauw
LUMC
Dept. of Anatomy & Embryology
Albinusdreef 2, S1-P
2333 ZA Leiden
The Netherlands
M.J.P.Nauw@lumc.nl
Christoph Schaniel PhD
Mount Sinai School of Medicine
One Gustave Levy Place, Box 1496
New York, New York 10029
USA christoph.schaniel@mssm.edu
Prof. Tsvee Lapidot
Weizmann Institute
Dept. Of Immunology
Rehovot, PO Box 26
7100 Israel
Tsvee.Lapidot@weizmann.ac.il
Prof. Ulrich Martin
MHH, LEBAO
Carl-Neuberg-Str. 1, HBZ, I11, H0
30625 Hannover
Germany martin.ulrich@mh-hannover.de
Sylvia Merkert MSc
MHH, LEBAO
Carl-Neuberg-Str. 1, HBZ, I11, H0
30625 Hannover
Germany merkert.sylvia@mh-hannover.de
Dr. Harald Mikkers
LUMC
Dept. of Molecular Cell Biology
Albinusdreef 2, S1-P
2333 ZA Leiden
The Netherlands h.mikkers@lumc.nl
Dr. Eduardo Moreno
CNIO
Melchor Fernandez Almagro 3
Madrid 28029
Spain emoreno@cnio.es
Dr. Catherine Robin
Erasmus Stem Cell Institute
Dept. Of Cell Biology
P.O. Box 2040
3000 CA Rotterdam
The Netherlands c.robin@erasmusmc.nl
Sunita L. D'Souza PhD
Mount Sinai School of Medicine
1425 Madison Avenue, Rm # 13-21
New York, New York 10029
USA dsouzs01@mssm.edu

Prof. Toshio Suda
Keio University School of Medicine
35 Shinanomachi, Shinjuku-ku
160-8582, Tokyo
Japan sudato@sc.itc.keio.ac.jp
ORGANIZING COMMITTEE
Dr. Gijs van den Brink
LUMC
Dept. of Gastroenterology and
Hepatology
Albinusdreef 2, C4-P
2333 ZA Leiden
The Netherlands
G.R.van_den_Brink@lumc.nl
Prof. Christine Mummery
LUMC
Dept. of Anatomy and Embryology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
C.L.Mummery@lumc.nl
Prof. Frank Staal
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
F.J.T.Staal@lumc.nl
Prof. Adrian Thrasher
UCL Institute of Child Health
30 Guilford Street
London WC1N 1EH
United Kingdom
A.Thrasher@ich.ucl.ac.uk
Prof. Wim Fibbe
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
W.E.Fibbe@lumc.nl
Dr. Melissa van Pel
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
M.van_Pel@lumc.nl
Dr. Jaap Jan Zwaginga
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
J.J.Zwaginga@lumc.nl