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.

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