The Niche

Last ISSCR 2009 post: thanks to many and congratulations to the poster winners

This month marked the 7th ISSCR meeting for the society and the third for me, and as always, there was much more news worth covering than bandwidth to do so.

I had some help this year, the result of an experiment asking for volunteer writers to send me summaries of the sessions and posters they found most interesting. These posts (you can find them by searching ISSCR or “conference blogs”) made the coverage this year fuller and wiser. I am very grateful for the contributions of Julie Clark, Andrea Ditadi, and Teisha Rowland. Also, thanks to professor Jeanne Loring for her comments on the write-up of Yamanaka’s talk, showing that iPS cells derived from different cell types behave differently.

If a dark-haired 30-something woman sidled up to you in Barcelona with an onslaught of questions (“What was your favorite session? Did you believe that last talk? Is X an established concept or a new idea?”), that was probably me. Thanks everyone for patiently sharing your knowledge, tips and insights. You literally make my job worth doing.

I also know there is a lot more to do. As we walked out of the last talk on the last day of the conference, I asked the outgoing society president Fiona Watt for some of her clearest memories of the past year. She was cheery at the conclusion of a meeting that she’d felt had gone well (our conversation was continually interrupted by attendees offering her handshakes and congratulations). Still, one of her comments struck me as a growing problem:

“Things obvious to one section of our community are not obvious to others.”

Finally, though I had nothing to do with it, I know several scientists put a lot of time into assessing and picking poster winners. I’m sure you’ll see some of this work in the peer-reviewed literature before too long. In the meantime, here’s a sneak peak.


The abstracts of the winning posters are pasted below this list. You can see all of Thursday’s poster abstracts here (odd numbers) and Friday’s here (even numbers)

DONG WOOK HAN, MAX PLANCK INSTITUTE FOR MOLECULAR BIOMEDICINE, GERMANY, FOR THE POSTER: SOX2 BOOSTS PLURIPOTENCY OF MOUSE EPIBLAST STEM CELLS (#936)

MAROUN KHOURY, KOCH INSTITUTE FOR INTEGRATIVE CANCER RESEARCH AT MIT, USA AND SMART IRG INFECTIOUS DISEASES, SINGAPORE, FOR THE POSTER:

ANGIOPOIETIN-LIKE-5 SECRETED BY MESENCHYMAL STEM CELLS SUPPORTS A LONG TERM EX VIVO EXPANSION OF HUMAN HEMATOPOIETIC STEM CELLS (#1401)

REINHOLD J. MEDINA, CENTRE FOR VISION & VASCULAR SCIENCE, QUEEN’S UNIVERSITY BELFAST, UNITED KINGDOM, FOR THE POSTER: HUMAN BLOOD-DERIVED ENDOTHELIAL PROGENITOR CELLS PROMOTE VASCULAR REPAIR IN THE ISCHAEMIC RETINA (#1524)

JONATHAN A. NOWAK, THE ROCKEFELLER UNIVERSITY, USA, FOR THE POSTER:

MOUSE HAIR FOLLICLE STEM CELLS ARE SPECIFIED EARLY AND GOVERN SKIN MORPHOGENESIS (#1053)

MARTA WALASEK, UNIVERSITY MEDICAL CENTER GRONINGEN, NETHERLANDS FOR THE POSTER: VALPROIC ACID AND LITHIUM POSTPONE MOUSE HEMATOPOIETIC STEM CELL DIFFERENTIATION IN EX VIVO CULTURES WITH GROWTH FACTORS (#1539)

HEIKO WURDAK, THE SCRIPPS RESEARCH INSTITUTE, USA, FOR THE POSTER: A NEUROGENIC SMALL MOLECULE ACCELERATES NEURONAL DIFFERENTIATION IN THE ADULT RAT DENTATE GYRUS (#306)

SOX2 BOOSTS PLURIPOTENCY OF MOUSE

EPIBLAST STEM CELLS

Han, Dong Wook, Do, Jeong Tae, Tapia, Natalia, Joo, Jin Young, AraAozo-

Bravo, Marcos J., Bernemann, Christof, Greber, Boris, Stehling, Martin,

Sterneckert, Jared, Schoeler, Hans R.

Department of Cell and Developmental Biology, Max Planck Institute for Molecular

Biomedicine, Muenster, Germany

Pluripotent EpiSCs, one type of epiblast-derived pluripotent stem cell, have

been newly established, but their pluripotential capabilities have not been

adequately defined. Thus, in the current study, we elucidated the pluripotential

capacities of EpiSCs by investigating their reprogramming potential using

a cell fusion protocol. While the fusion of ES cells and F9 EC cells resulted in

the efficient production of fusion hybrid colonies, epiblast-derived pluripotent

stem cells (EpiSCs) and P19 EC cells showed extremely low and delayed reprogramming

patterns. The low reprogramming potential of these cells could

be rescued by overexpressing Sox2, which is underexpressed in both EpiSCs

and P19 EC cells. Sox2 overexpression also resulted in a reduction in the time

required for reprogramming as well as in the enhancement of epigenetic modifications

of the hybrid cells, exemplified by DNA demethylation of Oct4 regulatory

regions and reactivation of the X chromosome. Sox2-overexpressing F9

cells, P19 EC cells, and EpiSCs all showed compact ES-like morphology and

the capability to proliferate under ES culture conditions for many passages.

Moreover, EpiSCs, which very rarely form chimeras, were capable of efficiently

forming chimeras and contributing to germ cell formation following Sox2

overexpression. Therefore, Sox2 overexpression alone is sufficient to dramatically

rescue the restricted pluripotential capacities of mouse epiblast-derived

pluripotent stem cells.

ANGIOPOIETIN-LIKE-5 SECRETED BY

MESENCHYMAL STEM CELLS SUPPORTS A

LONG TERM EX VIVO EXPANSION OF HUMAN

HEMATOPOIETIC STEM CELLS

Khoury, Maroun1, Drake, Adam2, Leskov, Ilya2, Fragoso, Maria2, Lodish,

Harvey3, Chen, Jianzhu1

1Koch Institute for Integrative Cancer Research at MIT and SMART IRG Infectious

Diseases, Massachussetts Institute of Technology and Singapore MIT Alliance for

Research and Technology, Cambridge and Singapore, MA, USA, 2Koch Institute

for Integrative Cancer Research at MIT, Massachussetts Institute of Technology,

Cambridge, MA, USA, 3Whitehead Institute for Biomedical Research and Department

of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

Bone marrow transplantation, hematopoietic gene therapy approaches, as

well as basic human hematopoiesis research are often limited by the numbers

of available hematopoietic stem cells (HSC). Hence, robust methods for long

term ex vivo expansion are required. Mesenchymal stem cells (MSC) play an

important role in supporting HSC by producing cytokines, growth factors,

and cell adhesion molecules involved in hematopoiesis. Angiopoietic-like-5

(Angptl5) is a recently identified factor that results in dramatic ex vivo

expansion of human HSC. In this study, we evaluate the efficiency with

which MSCs engineered to express Angptl5 can support ex vivo expansion

of umbilical cord blood-derived HSCs. Methods: HSCs were cocultured with

MSC-Angptl5 at a ratio of 1:5 in a serum free-media. Cultured cells were

analyzed for the expression of stem cell markers (CD34 and CD133). After 10

days of culture, human stem/progenitor cells were injected into sublethally

irradiated NOD/SCID/IL2Rgammanull newborn mice. Human cell engraftment in the blood and other tissues of the recipients was determined 14 weeks

later. Results: After 10 Days of culture, HSCs co-cultured with MSC-Angptl5

showed a 220-fold increase (FI) of total nucleated cells (TNC) and a 64 FI of

CD34+CD133+ (double positive cells, DPC). HSCs co-cultured with control

MSCs or in cell-free cultured media containing recombinant Angptl5 showed

a lower expansion (110 and 100 FI of TNC, 28 and 20 FI of DPC, respectively).

Moreover, we were able to maintain the co-culture expansion for more than

40 days, which resulted in a 12000 FI of DPC. Mice injected with expanded

cells from different culture conditions showed similar percentage of human

CD45+ cells (leukocytes) in the blood, spleen, and bone marrow at 14 weeks

post-transplantation. However, a higher absolute and relative number of T

cells and myeloid cells was observed in all 3 tissues of mice injected with

HSC co-cultured with MSC-Angptl5 when compared to those cultured in cellfree

media. Furthermore, the analysis of the engrafted CD45+ cells 6 months

after engraftment showed that both conditions were capable of a long-term

lymphomyeloid reconstitution. Conclusion: These results indicate that under

our optimized culture conditions, the HSC expansion can take place without

compromising the short or long-term repopulating properties of HSCs.

The method of HSC co-culture with MSCs that express Angptl5 and other

cytokines may pave the way for ex vivo expansion of human transplantable

HSCs suitable for clinical applications.

HUMAN BLOOD-DERIVED ENDOTHELIAL

PROGENITOR CELLS PROMOTE VASCULAR REPAIR

IN THE ISCHAEMIC RETINA

Medina, Reinhold J.1, O’Neill, Christina L.1, Humphreys, Mervyn W.2,

Gardiner, Tom A.1, Stitt, Alan W.1

1Centre for Vision & Vascular Science, Queen’s University Belfast, Belfast, United

Kingdom, 2Northern Ireland Regional Genetics Centre, Belfast City Hospital Trust,

Belfast, United Kingdom

Increasing evidence suggests a beneficial role for Endothelial Progenitor

Cells (EPCs) in revascularisation of ischaemic tissues. However, the precise

phenotypic nature and biologic function of these cells is not well defined;

therefore multiple populations have been collectively named EPCs. Recently

we have isolated and characterised a distinctive human blood-derived EPC

with high proliferative capacity and intrinsic tubulogenic potential, known

as Outgrowth Endothelial Cells (OECs). The aim of this study was to evaluate

the angiogenic potential of OECs in vitro and in vivo. OECs were isolated from

human peripheral blood or cord blood and propagated on collagen substrate.

The molecular phenotype of OECs was characterised by flow cytometry and

immunocytochemistry. Clonogenic and proliferative potential and senescence

analysis demonstrated that OECs could be single-cell cloned and reach over

60 population doublings in 70 days after which they exhibited a senescent

phenotype although they maintained a normal diploid karyotype. Tubulogenesis

assays demonstrated that OECs form a typical tube-like network when

cultured in Matrigel. OECs co-cultured with primary microvascular endothelial

cells formed a confluent uniform cell monolayer. Immunostaining for

cadherin, b-catenin and ZO-1 demonstrated that OECs interacted closely with

mature endothelial cells by forming adherens and tight junctions. Using an

in vitro 3D-angiogenesis model we found that OECs fully incorporated into a

microvascular tube network. For a parallel in vivo investigation, we utilised a

murine model of ischaemic retinopathy in which central retinal hypoxia drives

a pathologic “pre-retinal” neovascular response. OECs were labelled with

intracellular Qdots and delivered into the vitreous of mice with ischaemic

retinas. After 72 hours, confocal microscopy revealed that OECs integrated

into the “intra-retinal” microvasculature where they significantly contributed

to vascular repair by decreasing ischaemia (p<0.001), increasing the normovascular area (p<0.001) and thereby reducing the stimulus for pathological neovascularisation (p<0.01). In conclusion, OECs are a distinct endothelial progenitor cell subpopulation with the capacity to interact with mature endothelial cells and directly contribute to repair of ischaemic retina. OECs show considerable promise towards an exciting and novel cell-based therapy approach to induce therapeutic angiogenesis of ischaemic tissues. MOUSE HAIR FOLLICLE STEM CELLS ARE SPECIFIED EARLY AND GOVERN SKIN MORPHOGENESIS Nowak, Jonathan A., Polak, Lisa, Pasolli, Amalia, Fuchs, Elaine Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY, USA In adult skin, hair follicles undergo cyclic bouts of hair growth followed by sequential destructive and resting phases. This process is fueled by epithelial stem cells which reside in a quiescent niche associated with each hair follicle. In a wound environment, these stem cells can also be mobilized to regenerate the interfollicular epidermis and sebaceous glands. The architecture of the stem cell niche only becomes pronounced postnatally at the start of the first hair cycle. Whether stem cells exist or function earlier is unknown. By conducting in vivo pulse-chase studies of skin epithelial morphogenesis, we demonstrate that quiescent cells expressing numerous stem cell markers appear early in skin development, when hair follicles are still being actively established. These cells then later give rise to the adult stem cell population. To test whether these early quiescent follicle cells function as stem cells, we use Sox9-Cre for genetic marking and K14-Cre to embryonically ablate Sox9, an essential adult stem cell gene. We find that the progeny of Sox9- expressing cells contribute to all skin epithelial lineages and that adult hair follicles are completely derived from Sox9-expressing early stem cells. In the absence of Sox9, the early stem cell population is never specified. As a result, hair follicle and sebaceous gland morphogenesis is blocked and epidermal wound repair is severely compromised. These surprising findings establish the existence of early hair follicle stem #1539 VALPROIC ACID AND LITHIUM POSTPONE MOUSE HEMATOPOIETIC STEM CELL DIFFERENTIATION IN EX VIVO CULTURES WITH GROWTH FACTORS Walasek, Marta, van den Boom, Vincent, Dethmers-Ausema, Bertien, de Haan, Gerald, van Os, Ronald Cell Biology, University Medical Center Groningen, Groningen, Netherlands Hematopoietic stem cells (HSCs) are an atrractive source for many clinical stem cell therapies. Despite the fact that they are one of the best characterised stem cell in the body, the maintenance and expansion of HSCs in vitro remains a major challenge. Several clinical applications are hampered by difficulties in potent ex vivo expansion of HSCs, in which one of the major problem is concominant differentiation during cell proliferation. To maintain both stem cell functionality in vitro, the proliferation and differentiation of HSCs should be carefully controlled. Therefore, ex vivo expansion protocols should not only provide potent proliferation to increase the number of cells, but should also limit the differentiation of the stem cells. As inhibitors of stem cell differentiation we have chosen lithium (Li), a potent GSK-3b inhibitor (activating Wnt signaling) and valproic acid (VPA), an inhibitor of histone deacetylases. In this study we assessed the capacity of Li and VPA to modulate murine stem cell differentiation stimulated with strong differentiation signals, Stem Cell factor (SCF) and Granulocyte/ Macrophage-Colony Stimulating Factor (GM-CSF). Treatment with Li and VPA resulted in delay of murine HSCs differentiation in 7 days liquid cultures, preserving the immaturity of the cells, as shown by an increase in blast-like cell morphology and stem cell (Lin-Sca1+c-kit+) immunophenotype. Li and VPA strongly preserve expression of Sca1 marker, which resulted in 10-fold increase of LSK phenotype in treated vs control cells. Treatment with Li/VPA also increased functional activity of treated vs control cells, when measured by short-term and long-term stem cell in vitro activity (CFCs and CAFC) and in vivo activity. Transplantation experiments showed approximately 3-fold increase in competitive repopulation activity of cells treated with Li and VPA 12 weeks after transplantations. Gene expression analysis of cultured cells revealed that treatment with Li and VPA caused a significant upregulation of two genes relevant for stem cells : p57 and HoxA9, as well as an upregulation of p16 . To further study the molecular mechanism of the observed effect, and as VPA is a well known epigenetic modifier, we performed chromation immunoprecipitation (ChIP) assays to assess whether gene expression data correlate with the epigenetic status. ChIP assays showed significantly increased levels of H3 and H4 acetylation with concominant decrease in H3K27Me3 on the promoters of p16, p57 and HoxA9 in cells treated with Li and VPA (see abstract V. van den Boom). This data indicate that Li and VPA represent potent stem cell differentiation inhibitors, maintaining stem cell phenotype by preservation of a HSC-specific epigenetic profile. We suggest that lithium and valproic acid present two independent, but synergistic ways to stimulate hematopoietic stem cell growth while preventing differentiation. This research may contribute to a better understanding of the biology of HSCs in culture and may help to improve future application of ex vivo stem cell expansion protocols for clinical purpose. A NEUROGENIC SMALL MOLECULE ACCELERATES NEURONAL DIFFERENTIATION IN THE ADULT RAT DENTATE GYRUS Wurdak, Heiko1, Zhu, Shoutian1, Aimone, Lindsey2, Weerapana, Eranthie1, Lee, Jong Seok1, Chopiuk, Gregory2, Min, Kyung Hoon1, Zhang, Jay2, Walker, John2, Cravatt, Benjamin F.1, Gage, Fred H.3, Cho, Charles4, Schultz, Peter G.1 1The Scripps Research Institute, La Jolla, CA, USA, 2Genomics Institute of the Novartis Research Foundation, La Jolla, CA, USA, 3The Salk Institute for Biological Studies, La Jolla, CA, USA, 4Genomics Institute of the Novartis Research Foundation, La Jolla, CA, USA Neurogenesis in the adult brain occurs throughout life at two distinct locations: the Subventricular Zone, and the hippocampal Dentate Gyrus. These neurogenic niches harbor self-renewing neural progenitor cells which have the capacity to generate neurons, astrocytes and oligodendrocytes. However, little is known about signals controlling the balance of adult neural progenitor self-renewal and differentiation, and whether these signals can be pharmacologically modulated in order to improve brain tissue regeneration. We previously have carried out a phenotypic screen which identified a class of 4-aminothiazoles (termed KHS compounds), which strongly induces the selective neuronal differentiation of cultured rat neural progenitor cells. An SAR study afforded the molecule KHS101 which has reasonable half-life, therapeutic index and brain levels. Initial in vivo experiments with rats were performed to assess the effect of KHS101 on adult neurogenesis. Strikingly, the number of BrdU/NeuN-positive cells, indicative of neuronal differentiation in vivo, was significantly increased in the dentate gyrus of KHS101-treated rats compared to vehicle-treated animals. Target identification of KHS101 using affinity chromatography revealed specific binding to the centrosomal protein TACC3. TACC3 has been implicated in maintenance of HSCs and NSCs and may also be involved in mechanisms of deregulated self-renewal leading to tumorigenesis. Overall, these findings strongly suggest that KHS101 accelerates neuronal differentiation of adult neural progenitor cells by a novel mechanism and may provide a basis for modulating adult neural progenitor cells, ultimately in a therapeutic setting.

Comments

Comments are closed.