Stem

cells and regenera.on

Professor Mar.n benjamin.mar.n@stonybrook.edu Oce hours Fridays 3:30-5:00 480 LSB

Lecture outline
Stem cells in your body Maintaining and dieren.a.ng stem cells Nuclear reprogramming Stem cells and regenera.on

Stem cells: cells dened by their ability to self renew and dieren7ate into one or many mature cell types. Vertebrate stem cells: Embryonic stem cells Adult stem cells: Skin, hair follicles, Intes7ne, germ cells, hematopoei7c, heart, hippocampus Plant stem cells: Shoot apical meristem, root apical meristem ,oral mersitem Stem cell terminology: To7-potent, pluripotent, mul7-potent, etc. What do these terms mean?

Stages of early mammalian development

Embryonic stem cells are derived from the inner cell mass of the blastocyst

You started as a to.potent cell

As development proceeds, cells become more and more restricted in fate. To.potent stem cell gives rise to any cell type, including extraembryonic .ssue Pluripotent stem cell gives rise to all three germ layers Mul.potent stem cell gives rise to mul.ple restricted cell types There is such a thing as a unipotent stem cell only gives rise to one cell type

Classic view of germ layer forma.on and cell fate determina.on

Historical data led to a model of three germ layers being formed during gastrula.on Each germ layer gives rise to germ layer specic cell fates Recent data suggest that this model needs to be updated to incorporate a stem cell model of axial growth
Muscle, bone, heart, kidneys, etc. Gut, liver, lung, etc.

Skin, nervous system

Early development of the zebrash embryo

(by Rolf Karlstrom and Don Kane; Development 123:461, 1996)

Posterior growth and formation of the ! body! This embryo has already
nished gastrulation!

3-somite!

6-somite! 18-somite! younger! older! 31-somite!

Vertebrates grow from the neck to the posterior!

Two prevailing models regarding the state of tailbud progenitor/stem cells!

Classic model Neural progenitors! Neural progenitors! Pluripotent stem cells!
Notochord! Notochord!

Mesodermal progenitors! Pasteels xed fate model (1937)!

Mesodermal progenitors! Holmdahl blastema model (1925)!

Gene.c labeling of clonal cell popula.ons in the mouse embryo

Mice transgenic for an inac.ve LacZ gene are used for lineage labeling Rare mito.c recombina.on events create ac.ve LacZ Based on known cell division rate and clone size, developmental .me of recombina.on event can be extrapolated

Dev Cell. 2009 Sep;17(3):365-76

Clonal analysis indicates that the classic germ layer model does not hold true
Mice transgenic for an inac.ve LacZ gene are used for lineage labeling Rare mito.c recombina.on events create ac.ve LacZ Based on known cell division rate and clone size, developmental .me of recombina.on event can be extrapolated Neural and mesodermal .ssue are more closely related than neural and epidermal .ssue
Dev Cell. 2009 Sep;17(3):365-76

Posterior (tailbud) cells can self renew over extended periods

Posterior growth in embryos eventually stops. Does this mean that the cells required for posterior growth are not stem cells? Serially transplanted cells con.nue to divide and populate the body axis within neural and mesodermal .ssue types.

Development. 2002 Oct;129(20):4855-66.

How do stem cells know how to choose specic fates?

During body forma.on, Wnt signaling allocates tailbud stem cells to proper fates. Wnt signaling ini.ally species mesoderm over spinal cord. Wnt signaling controls a second fate decision within the mesoderm causing cells to become muscle rather than blood vessels.
Developmental Cell 22, 223232, January 17, 2012

Revised model of embryonic germ layer forma.on

The tradi.onal model of germ layer forma.on and its derivi.ves does not hold true for posterior .ssues. Paraxial mesoderm and neural plate are more closely related than neural plate and epidermis. Wnt signaling turns on Tbx6 expression and represses Sox2 expression.
Current Opinion in Gene.cs & Development 2012, 22:374380

Stem cell niches

What does the existence of an adherens junc7on imply for the regula7on of stem cells in simple or complex niches?

Stem cells are found in niches in the ovary, tes7s, bone marrow, intes7ne, skin, hair follicles, the heart and hippocampus. [a] In a simple niche a stem cell has a permanent partner to which it is aKached by an adherens junc7on. The cells divide asymmetrically to give another stem cell and a dieren7ated cell. [b] In a complex niche two or more dierent stem cells [red and pink] are supported by one or more partner cells. [c] In a storage niche stem cells are stored un7l ac7vated by an external signal.

Wnt signaling in stem cell niches

In HSC niche, Wnt signaling promotes stem cell prolifera.on. In hair follicle, Wnt signaling promotes transit amplica.on of dieren.a.ng cells.

Nature Reviews Molecular Cell Biology 9, 11-21 (January 2008)

The hematopoie.c stem cell

True pluripotent stem cells characteris7cs: 1. Pluripotent: capable of forming a mul7plicity of 7ssues. 2. If injected into an irradiated mouse [whose stem cells are destroyed]: A. Should migrate to bone marrow B. Should rescue mouse; stem cell proper7es restored. C. Cells from rescued mouse should rescue other irradiated mice. D. Should be capable of forming a mul7plicity of 7ssues in subsequent rescued mice.

xxxxxxxx

A true stem cell should rescue an irradiated mouse whose own bone marrow is destroyed.

The cell cycle in hematopoie.c stem cells

Typical of aging stem cells, HSCs go from 100% cycling in early life to nearly 100% quiescent in late life. JCB vol. 195 no. 5 709-720

Cell cycle control in hematopoie.c stem cells

Entry into G1 from G0 is regulate by compe.ng signals.

JCB vol. 195 no. 5 709-720

Cancer within the HSC lineage

Normal hematopoiesis and Chronic myelogenous leukemia.

N. Lobo ,Y. Shimono ,D. Qian & M.Clarke. Annual Review Cell & Developmental Biology 22: 675-699.2007

Mechanisms leading to cancer stem cell forma.on

Satellite cells are muscle stem cells

Satellite cells are found beneath the basal lamina surrounding muscle bers. Amer injury satellite cells are ac.vated and form new muscle.

Symmetric and asymmetric division of satellite cells

Transcrip.on factor hierarchy regula.ng muscle dieren.a.on

Transcrip.on factor expression regulates the transi.on from stem cell to dieren.ated muscle. Recall MyoD is a transcrip.on factor that turns on CKIs to halt the cell cycle.

Cold Spring Harb Perspect Biol 2012

Myosta.n regula.on of myogenesis from muscle stem cells

Myosta.n is a TGF- family member that inhibits myoblast prolifera.on and dieren.a.on.

Naturally occurring Myosta.n muta.ons

Hair follicle and intes.nal crypt

The dermal papilla (DP) signals to matrix stem cells (red) located across a basement membrane (green). Matrix cell daughters (yellow) dieren.ate into a variety of cell types, including the medulla, cortex and cu.cle of the hair sham (brown), the inner root sheath (IRS) and the outer root sheath (ORS). About two-thirds of the way up an anagen follicle lies the bulge an expanded region that contains long-term stem cells (red). These cells periodically replenish (arrows) the matrix cells, and also help maintain the sebaceous gland (SG) and the epidermal stem cells (red, top layer) that lie against the basement membrane (not shown) overlying the basal layer in interfollicular regions.

Wnt is involved in transit amplifying step

Nature 414, 98-104(1 November 2001)

Hair follicle stem cells can be used to grow new hair

Plants have stem cell too!

Growing root .p Experimental data calls in to ques7on the func7on of root 7p stem cell niche. Organ regenera7on does not require a func7onal stem cell niche in plants

Maintenance and dieren.a.on of embryonic stem cells

LIF and BMP signaling, along with pluripotency transcrip.on factors (Oct4, Sox2, and Nanog) maintain self-renewal FGF signaling promotes dieren.a.on. Self-renewal is maintained by ac.vely inhibi.ng dieren.a.on. If you allow ES cell cultures to reach high density, FGF builds up and LIF and BMP become depleted. Cells spontaneously dieren.ate.

Cell cycle control in stem cells

ES cells have cons.tu.vely ac.ve CDK2/cyclinE. This allows rapid progression in to S phase with a very short G1 (essen.ally no restric.on point) In fetal stem cells, mitogens ac.vate CDK4/6/ cyclinD which cooperates with CDK2/cyclinE for rapid S phase entry. Hmga2 represses CKIs.

Cell cycle control in stem cells

Young adult stem cells are mostly quiescent. Mitogen ac.vates them to enter the cell cycle. Old stem cells increase let7 microRNA expression which inhibits Hmga2. CKIs are expressed and it is more dicult for these cells to enter the cell cycle.

Lecture outline
Stem cells in your body Maintaining and dieren.a.ng stem cells Nuclear reprogramming Stem cells and regenera.on

Mechanisms suppressing stem cell dieren.a.on

RNA binding proteins block dieren.a.on specic transcripts Repression of dieren.a.on inducing genes by pluripotent transcrip.onal repressors Epigene.c modica.ons

Mechanisms suppressing stem cell dieren.a.on

lineage priming maintains undieren.ated state Asymmetric division segrega.ng dieren.a.on or stem cell factors Asymmetric division so that one cell remains in the stem cell niche

Niche regula.on of stem cell renewal

Drosophila germ stem cells are anchored to the niche by adherens junc.ons. This orients asymmetric cell division and keeps stem cell in pluripotent signaling environment. C. elegans germ cells ac.vated by Notch signaling. Notch ac.vates RNA binding proteins FBF-1 and FBF-2 which inhibit meiosis.

FoxO is involved in stem cell self- renewal

FoxO inac.va.on in cancer

Chroma.n changes associated with lineage specica.on

Oct4, Sox2, and Nanog are expressed in pluripotent cells. Repressed lineage restricted factors marked by bivalent chroma.n domains.

TGF- signaling during stem cell dieren.a.on

TGF- signaling through Smad 2/3 and Smad 4 ac.vates transcrip.on of homeostasis and dieren.a.on genes. A recently discovered branch of the pathway involving TRIM33 Smad 2/3 complex is also required to recognize repressive marks on chroma.n of dieren.a.on genes.

TGF- signaling during stem cell dieren.a.on

FEBS Lesers 586 (2012) 19531958

Nuclear lamina structure and stem cell maintenance

How Human Embryonic Stem Cells are Derived

( 2006 Terese Winslow)

Manipula.ng stem cell gene expression with siRNA

Manipula.ng stem cell fate determina.on with extracellular signaling factors

Lecture outline
Stem cells in your body Maintaining and dieren.a.ng stem cells Nuclear reprogramming Stem cells and regenera.on

Nuclear reprogramming
Seminal experiment by John Gurdon showing that dieren.ated cells s.ll have all of the gene.c material necessary to create a whole animal AND that the nucleus can be reprogrammed by cytoplasmic factors.

Cloned Xenopus laevis frogs using albinism as a marker

Detailed method for cloning a frog bby nuclear transfer

Cloning of mammals by nuclear transfer

Do cloned animals prematurely age?

Telomeres shorten with age in soma.c cells. Does cloning an animal with a reprogrammed soma.c cell cause all cells to start with shorter telomeres? Dolly the sheep died at a premature age, was this due to a cloning specic event?

Reprod Biol Endocrinol. 2003; 1: 105.

cause adverse conditions, birds may migrate to 2. Geiser, F. & Ruf, T. Physiol. Zool. 68, 935966 (1995). ary to avoid them. However, many birds are seden- 3. Cossins, A. R. & Barnes, B. M. Nature 382, 582583 (1996). 4. Brigham, R. M. Physiol. Zool. 65, 457472 (1992). (Tb). tary and often rely on ephemeral, weather- 5. Reintertsen, R. E. Polar Res. 1, 269284 (1983). stain- dependent food sources so how do they 6. Krtner, G. & Geiser, F. Oecologia 123, 350357 (2000). dverse overcome periodic energy bottlenecks? 7. Krtner, G. & Geiser, F. J. Zool. Lond. 248, 501507 (1999). To answer this question, we investigated small urvive whether the Australian tawny frogmouth udy of (Podargus strigoides ; Fig. 1), a sedentary ted to bird which feeds mainly on arthropods, Ageing Nuclear transfer uses torpor in the wild. The study was 2,4,5), was performed over conducted from the Australian autumn to come 6 genera.ons. summer in an open woodland of Eucalyptus brief com and Acacia at 1,000 m altitude in a cool Telomere length temperate area ice have been cloned by nuclear on sequentially was measure by near Armidale, New South telomere shorten 13 Wales. We captured seven frogmouths and transfer into enucleated oocytes , Clone come of the clo southern b lot. only 12% of r fitted them with temperature-sensitive and here we describe the reiterative live-born clones Telomeres do not for donor nuclei transmitters (calibrated to the nearest 0.1 cloning of mice to four and six generations cannot be exclud C) weighing 3 g. All birds received an in two independent lines. Successive generkb shorten amer serial required into th 48.5 4 transfer on telom external backpack-style transmitter ations showed no signs of premature (long cloning (they may Teruhiko Wakay kb Kellie L. K. Tama 48.5 by gross behavioural ageing, as judged range) to measure skin temperature (Tskin), 12.2 even g et l onger!). D. Caroline Blan 8.6 parameters, and there was no evidence of and three birds had a second internal transBlanchard||, Atsu Tanemura, Mak 12.2 mitter (short range), to measure core Tb shortening of telomeres at the ends of chroAnthony C. F. Pe Peter Mombaert 8.6 an indicator of cellular normally and to determine TbTskin differentials, mosomes, Line A Line B *Department of An implanted under general anaesthesia. senescence in fact, these appeared to John A. Burns Scho Figure 1 Telomere lengths in successive generations (G1G5) of mice cloned from cumulus cells. Southern-blot analysis of terminal Hawaii, Honolulu, increase slightly in length. This increase is Transmitter signals were recorded at 10restriction-enzyme-cut fragments in five sequential generations shows that telomeres do not undergo incremental erosion in successive Department of Ce clonal generations. Genomic DNA isolatedgiven from peripheral-blood lymphocytes taken from representative from each generation Research, Kyoto Un surprising, that the number of animals mitotic min intervals for up to nine months6. was digested with the restriction enzyme Hin fI, resolved on a pulse field gel, transferred to a solid support and probed with a 5- PKyoto 606-8507, Ja greatly exceeds that ofof mice sexually All individuals entered torpor in winter: labelled (T AGdivisions ) oligonucleotide. Peripheral blood lymphocytes were sampled on the same day. Ages (in months) were: in line A, Life Sciences Divis donor, 18; G1, 16; G2, 14; G3, 12; G4, 9; G5, 9; in line B, G1, 15.5; G2, 13; G3, 11; G4, 9; G5, 7. Suffix numbers (G4-1 and G4-2, for Laboratory, Univer produced animals and that any deleterious Tb fluctuated around 3840 C during example) identify different pups of each generation. Cailfornia 94720, U ||Bekesy Laboratory effects of cloning might be expected to be activity and fell to about 36 C during the NATURE | VOL 407 | 21 SEPTEMBER 2000 was repeated with cumulus cells from adult assays designed to monitor signs of prema- Hawaii, Honolulu, in cloned mice. Our rest phase, with a lower limit of 34 C. On such as a decline in activity in Department of Ve G1 mice amplified as nucleus donors to sequentially produce the ture ageing,

Serial cloning of mice does not reveal shortening of telomeres over subsequent genera.on
Cloning of mice to six generations

M
Donor G1 G2

G1-2

G2-2

G3-1

G3-2

G4-1

G2-1

G4-2

G1-1

G4-1

G3

G4-2

G5

32

3 3

Methods of nuclear reprogramming

Nuclear transfer puyng the nucleus of a dieren.ated cell into an enucleated oocyte. This is how Dolly was made. Pioneered by Gurdon. Cell fusion fusing a dieren.ated cell with an embryonic stem cell. Reprogramming using dened transcrip.on factors. Pioneered by Yamanaka.

Experimental method for the discovery of induced pluripotent stem cells

Clever method using Fbx15 promoter driving Geo and G418 selec.on. What is G418? Induced cells change shape, grow indenitely, and express stem cell markers.

Reprogramming cells requires a mesenchymal to epithelial transi.on

During development, EMT leads to dieren.a.on

Current Opinion in Gene.cs & Development 2012, 22:423428

Progression past the restric.on point requires the Rb protein

If the Rb kinase is lost, Rb remains bound to E2F and cell division is stopped.

Active Rb protein inhibits E2F protein. Mitogen-activated G1-Cdk leads to the phosphorylation of Rb which inactivates it and releasing active E2F protein which promotes progression through the cell cycle.

Myc promotes the cell cycle and is a proto- oncogene

What are the consequences of a mutation either in G1-Cdk or in Rb? .

Reprogramming by cell fusion

Cells can be fused to form heterokaryons (cells with gene.cally dis.nct nuclei). Using dierent species allows tracking of cell-type specic DNA. Specic factors required for reprogramming can be iden.ed and then tested in other stem cell systems.

John Gurdons nuclear transfer experiments

Timeline of nuclear reprogramming studies

Dolly is cloned by nuclear transfer

Yamanakas induced pluripotent stem cells

Transdieren.a.on using a single transcrip.on factor

2012 Nobel Prize in Physiology or Medicine

Nuclear transfer Induced pluripotent stem cells (iPS).

John Gurdons high school science report card

Lecture outline
Stem cells in your body Maintaining and dieren.a.ng stem cells Nuclear reprogramming Stem cells and regenera.on

Animals can regenerate by 3 general mechanisms

Unipotent stem cells Pluripotent stem cells Stem cells

Dedieren.a.on

Transdieren.a.on

Developmental Cell 21, July 19, 2011

In general, more basal animals have higher regenera.ve capacity

Mammals, birds, and frogs have the least neural regenera.ve capacity.

Nature Reviews Neuroscience 10, 713-723 (October 2009)

Mechanisms of neural regenera.on

Where else can we look to learn more about regenera.on?

Hydra
Hydra Regenera.on Is Accomplished with Three Dierent Stem Cell Popula.ons(A) Hydra are cnidarians with a primary body axis containing a hypostome (or head) at one end and a foot at the other. Cell prolifera.on in the body column con.nually pushes cells to the poles of the body. Asexual reproduc.on is accomplished by budding.(B) The body wall contains two epithelial cell layers, ectodermal and endodermal epithelial cells. Inters..al stem cells exist within the ectodermal epithelial cell layer.(C) The ectodermal and endodermal epithelial cells proliferate con.nuously to maintain these .ssue layers, producing dieren.ated epithelial cells, and are therefore considered to be dis.nct stem cells. A third stem cell type, the mul.potent, inters..al stem cell can self-renew and produce neurons, nematocytes, secretory cells, and gametes.

Developmental Cell 21, July 19, 2011

Frog tail and salamander limb

Cell Tracking of GFP-Labeled Cells in Amphibians Shows that Vertebrate Appendage Regenera.on Occurs by Producing Lineage-Restricted Progenitors in the Xenopus Tail and Axolotl Limb BlastemaCell labeling was primarily achieved via graming of embryonic .ssues during the neurula stage from GFP-expressing donors to normal hosts. (Top) Xenopus: posterior neural plate, presomi.c mesoderm, and notochord were transplanted to label tail spinal cord, muscle, and notochord, respec.vely. Amer tail amputa.on, the labeled .ssues regenerated the same .ssue type as prior to amputa.on (Gargioli and Slack, 2004). (Bosom) Axolotl limb Schwann cells and muscle were labeled by embryonic presomi.c mesoderm and neural crest transplanta.on (Kragl et al., 2009). Dermis and car.lage were labeled by direct .ssue transplanta.on in the limb, as well as embryonic .ssue grams (Kragl et al., 2009). Amer limb amputa.on, labeled Schwann cells regenerated Schwann cells only. Muscle regenerated muscle and no car.lage. Dermis regenerated dermis, car.lage and connec.ve .ssues (also described by Dunis and Namenwirth [1977]), while car.lage regenerated car.lage (also described by Steen [1968]).

Developmental Cell 21, July 19, 2011

Zebrash heart and n

Cre/loxP-Based Cell Fate Mapping Establishes that Dedieren.a.on Occurs during Zebrash Heart and Fin Regenera.on(A) Prior to heart resec.on, cardiomyocytes were labeled via a cardiomyocyte-specic promoter driving CreER expression. CreER, which is ac.ve in the presence of 4-HT (4-hydroxytamoxifen), acted on a cardiomyocyte-specic loxP reporter to excise a oxed STOP cassese, resul.ng in GFP expression. Newly regenerated cardiomyocytes (right, below dosed line) express GFP, indica.ng that they derived from cardiomyocytes in the injured heart .ssue ( Kikuchi et al., 2010 and Jopling et al., 2010).(B) Tracking of osteoblasts during caudal n regenera.on demonstrates that they contribute to the regenerated n and remain restricted to an osteoblast iden.ty. osterix:Cre-ERT2 ac.ng on the loxP reporter; upon Cre-mediated excision of a STOP cassese, the hsp70 promoter drives expression of GFP. GFP expression was induced prior to n amputa.on leading to sporadic cell labeling. GFP-expressing cells generate osteoblasts in the regenerated n, indica.ng that osteoblasts dedieren.ated and divided to produce more osteoblasts, remaining restricted to the osteoblast fate during regenera.on (Knopf et al., 2011).

Developmental Cell 21, July 19, 2011

Planaria
Planarian Regenera.on Is Accomplished with Pluripotent Stem Cells Called cNeoblasts(A) Neoblasts (blue) are the soma.c dividing cells of planarians and are depicted in blue. Dividing cells are scasered throughout the body, but restricted to behind the eyes and absent from the pharynx (centrally located). (B) Irradia.on with 1750 rad can result in animals with a single surviving dividing cell. This single cell, a clonogenic neoblast (cNeoblast), can divide and produce a colony of dividing cells, ul.mately producing dieren.ated cells spanning germ layers (Wagner et al., 2011). For example, individual cNeoblasts can generate both neurons and intes.ne cells, as well as dened dividing cell progeny popula.ons.(C) Irradia.on with 6000 rad eliminates all dividing cells. Transplant of a single cNeoblast from a donor strain (red) results in clonogenic growth and, ul.mately, the restored capacity for regenera.on.

Developmental Cell 21, July 19, 2011

Planarian brain regeneration.

Gentile L et al. Dis. Model. Mech. 2011;4:12-19

2011 by The Company of Biologists Limited

Stem cells and regeneration in planaria.

Gentile L et al. Dis. Model. Mech. 2011;4:12-19

2011 by The Company of Biologists Limited

Using RNAi to screen for genes essen.al for regenera.on

Wnt signaling controls iden.ty of regenera.ng .ssue

Science 18 January 2008: vol. 319 no. 5861 327-330

iPS cells may be used for regenera.ve medicine

Nat Cell Biol. 2011 May; 13(5): 497505.

Self assembly of .ssues in culture

a, Fundamental dierence between self- organiza.on in physics and in mul.cellular systems. Compared with self-organiza.on in biological systems (shown here by the forma.on of an organ bud), whose elements change and local rules of interac.on evolve spa.otemporally, the elements and local rules in physics and chemistry systems (shown here as the forma.on of a snowake) are constant. Biological systems are also inuenced by s.gmergy the history dependency of a system; in this case s.gmergy aects the local rules of interac.on at mul.ple steps amer a cell aggregate forms. b, In biological systems mul.cellular self-organiza.on involves a combina.on of self-assembly, self-paserning and self-driven morphogenesis. Self-assembly involves the .me-evolving control of cell posi.ons rela.ve to each other, such as in the forma.on of a layered pasern in .ssue development. Self-paserning is the spa.otemporal control of cell status, so that cells acquire heterogeneous proper.es in a region-specic manner from a homegeneous cell popula.on. Self-driven morphogenesis is the spa.otemporal control of intrinsic .ssue mechanics, shown here as a representa.on of deforma.on of re.nal epithelium

Self organizing ES cells

a, Embryonic stem (ES) cell culture with three-dimensional aggregates for self- organiza.on, depending on the medium, into stra.ed cor.cal .ssue (top), and the op.c cup and the mul.layered neural re.na (middle) from homogenous progenitors. Other .ssues, however, require interac.on with dierent .ssues in order to self-organize. For example, Rathke's pouch (bosom), from which pituitary .ssue develops, forms from oral ectoderm under the inuence of hypothalamic neuroectoderm. b, Self- forma.on of crypt-like organoids from a single-cell culture of intes.nal stem cells that self-renew and produce cells such as Paneth cells and enterocytes, crea.ng a niche in which the organoid can develop. c, Teeth develop by conjuga.on of 3D reaggregates of oral ectoderm and tooth mesenchyme cultured in collagen gel to generate a tooth-germ structure, which can grow into a tooth amer transplanta.on into the host, such as mouse alveolar bone.

Signaling interac.on during re.nal development

Re.nal pigment epithelium (RPE) and the neural re.na inhibit each other's development through Wnt proteins which promote RPE dieren.a.on at the cost of neural re.na dieren.a.on and Dkk (a diusible Wnt antagonist). RPE reinforces its own fate by augmen.ng Wnt signals with increased expression of Wnt ligands and the transcrip.on factor Mi. Conversely, neural re.na stabilizes its fate as a result of Wnt inhibi.on by the Wnt antagonist Dkk and the Wnt ligand expression suppressor Six3. Chx10 species neural re.na fate, but is only indirectly related to Wnt signalling control. In addi.on, the neural re.na produces FGFs, which promote the .ssues dieren.a.on at the cost of RPE dieren.a.on. a, Representa.on of region-specic distribu.on of factors that inuence dieren.a.on of RPE and the neural re.na in the self-organizing op.c cup. b, A hypothe.cal rela.onship of mutual interac.ons between RPE and the neural re.na in ES cell culture.

Phases of self-driven morphogenesis in the op.c cup

Four phases of self-driven morphogenesis of the op.c cup in embryonic stem cell culture. The re.nal epithelium forms the op.c cup by evagina.on (phase 1), the distal epithelium becomes relaxed and asened (phase 2), the epithelial junc.on undergoes a strong apical constric.on (phase 3), and the neural re.na expands and invaginates to form an op.c-cup shape (phase 4). Three local rules for the .ssue mechanics involved have been elucidated40, 81: rule 1, mechanical relaxa.on of the neural re.na by local reduc.on of ac.ve myosin (loss of the phosphorylated regulatory subunit of myosin pMLC2 results in a loss of ac.ve myosin); rule 2, strong apical constric.on of the hinge epithelium (making this por.on of the epithelium wedge-shaped); and rule 3, rapid tangen.al expansion of the neural re.na, which causes inward bucking of the .ssue by genera.ng compression. Compression in the neural re.na was demonstrated by three-dimensional (3D) specic cell abla.on through pinpointed irradia.on with a mul.photon (MP) laser40, in which the gap lem by the abalated cell was quickly lled by strong lateral compression. How local mechanical rules for .ssue s.ness, strain and stress are self-controlled in a phase-specic manner is a fundamental ques.on for future studies in four-dimensional (4D) force biology.

Self driven morphogenesis of the op.c cup

Use natural scaolds to build organs

H. OK, T. MaKhiesen, S-K Goh, L. Black, S. Kren, T.Netho & D. Taylor. Nature Medicine 14:213-221. Feb. 2008

3D printers can be used to print organs using stem cells