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Stem Cells: Minireview Presentation

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Information about Stem Cells: Minireview Presentation

Published on March 20, 2009

Author: jme17

Source: slideshare.net

Description

PowerPoint giving a summary on research in stem cells (brief historical overview), and the explanatory component of the papers which changed the game of stem cell research Yamanka's Nuclear Reprogramming.
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Stem Cells Dr. Tai-ping Sun Biology 280S: Biotechnology & Genetic Engineering Jon Martin Joshua Mendoza-Elias Fall 2008

Outline: I. Introduction A. Definition of Stem Cell B. History and Discovery of SCs II. Therapeutic Applications of SCs A. Methods for studying Stem Cells B. Problems with cloning C. Alternatives to cloning III. Policy IV. Nuclear Reprogramming

I. Introduction

A. Definition of Stem Cell

B. History and Discovery of SCs

II. Therapeutic Applications of SCs

A. Methods for studying Stem Cells

B. Problems with cloning

C. Alternatives to cloning

III. Policy

IV. Nuclear Reprogramming

Properties of Stem Cells Self-renewal Potency ・ Totipotent : Includes fertilized zygote and first few divisions of the fertilized egg. These cells can differentiate into embryonic and extraembryonic cell types. ・ Pluripotent: SC’s are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. ・ Multipotent: SC’s can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). ・ Unipotent : SC’s cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).

Self-renewal

Potency

・ Totipotent : Includes fertilized zygote and first few divisions of the fertilized egg. These cells can differentiate into embryonic and extraembryonic cell types.

・ Pluripotent: SC’s are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers.

・ Multipotent: SC’s can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.).

・ Unipotent : SC’s cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells).

How do SC arise?

Where do SC come from? Embryonic Fetal Adult Berashis Stem Cells

When do SC arise?

For the love of cloning Nuclear transfer technique: Donor egg: oocyte from female denucleated Cloned: Nucleus from Blastula of albino frog transferred to donor egg. J.B. Gurdon (1962): S. African clawed frog ( Xenopus laevis ) Evidence from EvoDevoBio: The development of cloning techniques revealed organisms could be grown from one cell.

Nuclear transfer technique:

Donor egg: oocyte from female denucleated

Cloned: Nucleus from Blastula of albino frog

transferred to donor egg.

One of these things is not like the other. Evidence from immunology suggests that a cell type possesses similar totipotent Qualities of ES cells in adult cells. Proof of Concept: Clinical Procedure:

Evidence from immunology

suggests that a cell type

possesses similar totipotent

Qualities of ES cells in adult cells.

Mechanism of progenitor cells in the first steps of B-cell maturation. Receptors and ligands look familiar? ( c-kit , SCF )

Techniques for studying ES cells The more things change, the more the stay the same. Experimental techniques focus on determining “ uniqueness” of ES cells in the hopes of imitating it. -genetic expression profiles -Epigenetic state of genome -juxtracrine/endocrine signaling -transplantation effects -TF’s

Experimental techniques focus on determining

“ uniqueness” of ES cells in the hopes of

imitating it.

-genetic expression profiles

-Epigenetic state of genome

-juxtracrine/endocrine signaling

-transplantation effects

-TF’s

II. Techniques in Stem Cell Therapy

Therapeutic Cloning Nuclear Transfer: Method is the same as was used to clone Dolly the sheep in February 1997

Nuclear Transfer: Method is the same as was used to clone Dolly the sheep in February 1997

Therapeutic Cloning

Problems with Therapeutic Cloning Cost Destruction of embryos Inefficiency – 277 cells taken from Dolly’s “mother,” but only 30 became blastocysts. (13.21%)

Cost

Destruction of embryos

Inefficiency – 277 cells taken from Dolly’s “mother,” but only 30 became blastocysts. (13.21%)

Alternative Therapeutic Methods Immunosuppression Associated risks “ Invisible” cells Notch protein MHC replacement

Immunosuppression

Associated risks

“ Invisible” cells

Notch protein

MHC replacement

Notch Protein

Major Histocompatablility Complex

 

Other Alternative Methods Use of haematopoietic stem cells (HSCs) Own bone marrow

Use of haematopoietic stem cells (HSCs)

Own bone marrow

The Heart Bone marrow stem cells effective in trials to restore lost tissue Thigh tissue has also been used, though problems have arisen Use of mesenchymal stem cells and G-SCF

Bone marrow stem cells effective in trials to restore lost tissue

Thigh tissue has also been used, though problems have arisen

Use of mesenchymal stem cells and G-SCF

Study: Orlic et al. Studied effects of using bone marrow stem cells to treat mice that suffered heart attacks Sorted bone marrow cells according to whether they expressed c-kit , a surface protein found on HSCs Transplanted into heart

Studied effects of using bone marrow stem cells to treat mice that suffered heart attacks

Sorted bone marrow cells according to whether they expressed c-kit , a surface protein found on HSCs

Transplanted into heart

Orlic Study Results Arrows indicate regenerating myocardium VM: viable myocardium MI: myocardial infarcted Red = myosin. There is less in the infarcted region. Green = Nuclei Areas of growth indicated by arrows Figure 1(a) Magnification: 12x

Arrows indicate regenerating myocardium

VM: viable myocardium

MI: myocardial infarcted

Red = myosin. There is less in the infarcted region.

Green = Nuclei

Areas of growth indicated by arrows

Study Results, cont’d 68% of the space affected by heart attack was replaced by new myocardium New tissue had myocytes and vascular structures Improved haemodynamics over untreated heart ( c-kit negative)

68% of the space affected by heart attack was replaced by new myocardium

New tissue had myocytes and vascular structures

Improved haemodynamics over untreated heart ( c-kit negative)

III. Policy Issues No federal law criminalizing destruction of embryos, though some states have these laws Proposition 71 in California – explicitly permits research using somatic cell nuclear transfer 2008 presidential election could bring about a significant change in current policy What’s your opinion? Current policy only allows federal funding for existing embryonic stem cell lines (21 in all), not new ones

No federal law criminalizing destruction of embryos, though some states have these laws

Proposition 71 in California – explicitly permits research using somatic cell nuclear transfer

2008 presidential election could bring about a significant change in current policy

What’s your opinion?

Current policy only allows federal funding for existing embryonic stem cell lines (21 in all), not new ones

Stem Cell Viddeo http: //youtube .com/watch? v=mUcE1Y_bOQE

IV. A Transcriptional Logic for Nuclear Reprogramming Takahashi & Yamanaka/ Kit T. Rodolfa and Kevin Eggan Cell (2006) 126: 652-655  . Background: -Pluripotency: nuclear transfer or fusion with ES. -Factors for reprogramming unknown. Questions: (i.) Minimal factors? (ii.) Can we use make ES-like cells? (iii.) How do these cells compare to ES cells?

 . Background:

-Pluripotency: nuclear transfer or fusion with ES.

-Factors for reprogramming unknown.

Questions:

(i.) Minimal factors?

(ii.) Can we use make ES-like cells?

(iii.) How do these cells compare to ES cells?

Trans Reprogramming continued Hypothesis : (i.) We can find factors. (ii.) We can build pluripotent cells. Experimental Design: -MEF and Tail Tip Fibroblasts (TTF) introduce factors via retrovirus -Test cell cultures.

Hypothesis :

(i.) We can find factors.

(ii.) We can build pluripotent cells.

Experimental Design:

-MEF and Tail Tip Fibroblasts (TTF) introduce factors via retrovirus

-Test cell cultures.

Nuclear Reprogramming Continued The Experiment: Figure 1: Reprogramming Differentiated Somatic Cells Individual factors insufficient to trigger embryonic reporter (Fbx15:  -geo). 24 previously identified genes: Self-renewal : Oct3 /4, Sox2 , Nanog Pluripotency: c-myc , Eras , Klf4 Q: What’s the magic formula? Results: Iterate through combinations. Narrowed pool down to 4 cDNAs (all above except Nanog).

The Experiment:

Comparison of Methods

Nuclear Reprogramming cont’d: Results & Discussion Q3: How do iPS cells compare to ES cells?  iPS-TFF chimeric mice X No chimerism post natal animals. Clone-to-clone in expression variation. Certain genes “lost” Epigenetics: intermediate to somatic & ES cells.  . Future Directions: iPS cells different. Can additional reprogramming fix this?

Q3: How do iPS cells compare to ES cells?

 iPS-TFF chimeric mice

X No chimerism post natal animals.

Clone-to-clone in expression variation. Certain genes “lost”

Epigenetics: intermediate to somatic & ES cells.

 . Future Directions:

iPS cells different. Can additional reprogramming fix this?

Generation of germline-competent induced pluripotent stem cells Keisuke Okita, Tomoko Ichisaka, & Shinya Yamanaka Nature 448 : 313-317  . Previously . . . -Retroviral introduction Oct3/4 , Sox2 , c-Myc , and Klf4 -Fbx15 IPS cells similar to ES cells, but different: gene expression profile, epigenetics, & NO adult chimeras. Q: Can we build better ES-mimetic cells called Nanog iPS cell clones? H: You betcha! Using Nanog should make them germline-competent. E: Same idea but new trick. New GFP marker used to indicate pluripotency. Then, add the four previously identified factors. Screen/culture/test.

 . Previously . . .

-Retroviral introduction Oct3/4 , Sox2 , c-Myc , and Klf4

-Fbx15 IPS cells similar to ES cells, but different:

gene expression profile, epigenetics, & NO adult chimeras.

Q: Can we build better ES-mimetic cells called Nanog iPS cell clones?

H: You betcha! Using Nanog should make them germline-competent.

E: Same idea but new trick. New GFP marker used to indicate pluripotency. Then, add the four previously identified factors. Screen/culture/test.

Generation of germline. . . continued The Experiment: Construct : Enhanced GFP (EGFP)-internal ribosome entry site (IRES)-puromycin resistance (Pur R ) into 5’ UTR Results: Nanog iPS cells found in blastocyst, migrating primordial germ cells (9.5 dpc), & genital ridges (13.5 dpc). Teratomas generated with 3 germ layers. ~5% were were GFP positive - Cells indistinguishable form ES cells (morphology/proliferation*) Nanog iPS, Fbx15 iPS, and ES cells were then characterized.

The Experiment:

Results:

Nanog iPS cells found in blastocyst, migrating primordial germ cells (9.5 dpc), & genital ridges (13.5 dpc).

Teratomas generated with 3 germ layers.

Results Q: How sim/diff are Nanog iPS cells to Fbx15 iPS cells and ES cells? RT-PCR data: Nanog iPS cells expression profile closer to ES cell profile Epigenetics: Methylation signature closer to ES cells SSLP (single sequence length polymorphism): Unique signature* Induction Efficiency: Nanog iPS cells: 0.001-0.03% Fbx15 iPS cells: 0.01-0.5% Differentiation in LIF & RA of Nanog iPS more closely resembles ES cells.

Q: How sim/diff are Nanog iPS cells to Fbx15 iPS cells and ES cells?

RT-PCR data: Nanog iPS cells expression profile closer to ES cell profile

Discussion: Nanog iPS cells were “more ES-like” Germline competism: Chimerism: 10%-90% Cross: -Male mice had small testes and aspermatogenesis -No Nanog iPS cell in mature sperm -F 1 confirmed transmission of reporter • Germline competency Clinical Applications: - c-myc lead to tumour reactivation • Advise transient expression system Low induction efficiency: -Rare SC coexisting with MEF culture? • Other determinants

Nanog iPS cells were “more ES-like”

Germline competism:

Chimerism: 10%-90%

Cross:

-Male mice had small testes and aspermatogenesis

-No Nanog iPS cell in mature sperm

-F 1 confirmed transmission of reporter

• Germline competency

Clinical Applications:

- c-myc lead to tumour reactivation

• Advise transient expression system

Low induction efficiency:

-Rare SC coexisting with MEF culture?

• Other determinants

Conclusion ES cell analogues are possible Epigenetics Further refinement Identification of Factors Delivery system

ES cell analogues are possible

Epigenetics

Further refinement

Identification of Factors

Delivery system

References: [1] Aldhous, P. Can they rebuild us? Nature (2001) 410 : 622-625. [2] Wadman, M. Stem-cell issue moves up the US agenda. Nature (2007) 446 : 842. [3] Holden, C. California's proposition 71 launches stem cell gold rush. Science (2004) 306 : 1111. [4] Couzin, J. and Vogel, G. Renovating the heart. Science (2004) 304 : 192-194. [5] Orlic, D., Kajstura, J., Chimenti, Stefano. Jakoniuk, I., Anderson, S.M., Baosheng, L., Pickel, J., McKay, R., Nadal-Ginard, B., Bodline, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410 : 701-705. [6] Rodolfa, K.T., and Eggan, K. (2006) A transcriptional logic for nuclear reprogramming. Cell 126 : 652-655. [7] Okita, K., Ichisaka, T., and Yamanaka, S.. Generation of germline-competent induced pluripotent stem cells. Nature (2007) 448: , 313-317.

[1] Aldhous, P. Can they rebuild us? Nature (2001) 410 : 622-625.

[2] Wadman, M. Stem-cell issue moves up the US agenda. Nature (2007) 446 : 842.

[3] Holden, C. California's proposition 71 launches stem cell gold rush. Science (2004) 306 : 1111.

[4] Couzin, J. and Vogel, G. Renovating the heart. Science (2004) 304 : 192-194.

[5] Orlic, D., Kajstura, J., Chimenti, Stefano. Jakoniuk, I., Anderson, S.M., Baosheng, L., Pickel, J., McKay, R., Nadal-Ginard, B., Bodline, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410 : 701-705.

[6] Rodolfa, K.T., and Eggan, K. (2006) A transcriptional logic for nuclear reprogramming. Cell 126 : 652-655.

[7] Okita, K., Ichisaka, T., and Yamanaka, S.. Generation of germline-competent induced pluripotent stem cells. Nature (2007) 448: , 313-317.

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