Stem Cell Therapy - Nebraska Surgical Researchnebraskasurgicalresearch.com/s NSR/presentation_pdf/2006... · 2004 Murine parthenogenesis (Kono, Tokyo) 2004-5 Hwang misconduct (Seoul),

Stem Cell Therapy

Mark A. Carlson, MD

Department of SurgeryUniversity of Nebraska Medical CenterVeterans Administration Health Center

Omaha, Nebraska

October 18, 2006

Di$closures:

None

Stem Cell Therapy:Overview

• Purpose• Definitions & terminology• History• Current applications

Human embryonic stem cell colony

Purpose

What is the ultimate goal?Col

um

bia

Pic

ture

s, 1

975

Purpose

intact organism

compensatory hypertrophy

tissue loss

(pathologicprocess)

wound healing

EPIMORPHIC REGENERATION

Purpose

organism size

regenerativeability

organism complexity

Regeneration through the phyla

The larger and/or more advanced the animal, the less it can regenerate.

Hydra

H. simpson

High

1. Epidermis2. Liver3. Endothelium4. Epithelium

Low (or theoretically possible)

1. Everything else

Tissue-Specific Mammalian Regenerative Capacity

Purpose

What is a stem cell? Two, Maybe Three, Criteria

Definitions & Terminology

1. Self-renewal

Nat

Rev

Im

munol

2006



2. Differentiation into multiple cell types

Stem Cell Research Foundation



3. In vivo reconstitution of tissue*

Cel

l 2003

red = cardiac myosingreen = adult stem cells, derived from myocardiumblue = nucleimagenta = α-smooth muscle actin

murine LV

infarct

Stem cell potency

Totipotent Pluripotent Multipotent Progenitor

zygote embryonic stem hematopoietic stem intestinal crypt

MesodermEndodermEctoderm

and

GermTrophoblast

MesodermEndodermEctoderm

(i.e., entire organism)

Organ specific Cell specific

PLASTICITY


Unipotent/

Stem cell potency: a hierarchy


totipotent

pluripotent

unipotent

pote

ncy

entire organism

(all 3 embryonic layers+ germ cells)

zygote blastocyst Mus musculus

hESC

mesoderm

endoderm

ectodermmultipotent

HSC

intestinal crypt villus tip

lymphocyte

neutrophil

monocyte

platelet

erythrocyte

cardiac muscle

panc acinus

neuron

Stem cells: plasticity criteria

1. Differentiation into multiple lineages2. Differentiated cells are functional3. Engraftment in vivo is “robust & persistent”

For adult stem cells, plasticity is an issue…

A cautionary note on myocardial regeneration:


Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Balsam LB, WagersAJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Nature 2004;428:668.

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Murry CE,Soonpaa MH, Reinecke H, Nakajima H, Nakajima HO, Rubart M, Pasumarthi KB, Ismail Virag J, Bartelmez SH,Poppa V, Bradford G, Dowell JD, Williams DA, Field LJ. Nature 2004;428:664.

undifferentiated stem cell

differentiated daughter cell

forward pathway: canonical(differentiation)

reverse pathway = DEDIFFERENTIATION

(Der Alchemist, E. van Hove)


Stem cells & the dedifferentiation concept

• Does this pathway exist?• If so, where, how, and how frequently?

The amphibian limb: Dedifferentiation in nature?


Blastema formation:central event of limb regeneration

(Goss, 1992)


Dedifferentiation in nature?

Limb amputation blastema

?

What’s in there?

A. Stem cellsB. Dediff cellsC. A & B


Dedifferentiation in nature?

WHO CARES?



totipotent

pluripotent

unipotent

pote

ncy

entire organism



hESC

mesoderm

endoderm

ectodermmultipotent

HSC


lymphocyte

neutrophil

monocyte

platelet

erythrocyte

cardiac muscle

panc acinus

neuron

History

1938 Nuclear transfer techniques conceived (Spemann, Freiburg)

1943 DNA implicated as genetic material (Avery et al., NYC)

1952 Frog cloned with transfer of embryonic nucleus (Briggs & King, Philadelphia)

1953 Structure of DNA (Watson & Crick, Cambridge)

1958 Frog cloned with transfer of somatic nucleus (Gurdon et al., Oxford)

1960’s Adult neurogenesis (Altman & Das, MIT) 1963 Murine bone marrow stem cells (McCulloch & Till, Toronto)

1968 Bone marrow transplant (UMinn/UWisc)

1972 Recombinant DNA (Berg et al., Stanford)

1973 Moratorium on federal funding of research involving human embryos

1978 Human born after in vitro fertilization (IVF) in England

1981 House embryonic stem cells from the blastocyst (Evans & Kaufman, Cambridge; Martin, UCSF)

1984 Sheep cloned with transfer of embryonic cells (Willadsen, Cambridge)1993 Repeal of 1973 funding moratorium

1995 Dickey Amendment (no Federal $ for research involving embryo destruction)

1996 “Dolly” (sheep) cloned with transfer of somatic nucleus (Wilmut et al., Roslin Institute)

(continued)

Timeline

History

1998 Human embryonic stem cells from the blastocyst (Thomson et al., UWisc)

1998 Cloned mouse blastocyst (Mountford et al., Victoria, Australia)

2001 Federal funding allowed on existing embryonic stem cell lines

2001 Cloned human blastocyst* (Cibelli et al., Advanced Cell Technology)

2001 Murine cardiac regeneration with bone marrow cells (Anversa et al., NY Med Coll)*

2002 Derivation of pluripotent stem cells from murine bone marrow (Verfaillie et al., UMinn)

2003 Oocytes derived from mouse embryonic stem cells (Schöler et al., UPenn)

2004 Murine parthenogenesis (Kono, Tokyo)

2004-5 Hwang misconduct (Seoul), human ESC from nuclear transfer

2005 Embryonic stem cells without embryo destruction (Lanza et al., Advanced Cell Technology)

2006 Human embryonic stem cells from arrested “nonviable” embryos (Stojkovic, Spain)

2006 Oct4, Sox2, c-Myc, and Klf4 induce pluripotency (Takahashi & Yamanaka, Kyoto)

2006 Human embryonic stem cells from unfertilized eggs (Univ Milan, unpublished)

2006 Murine blastocyst from somatic nuclear transfer with cow oocyte (Seoul, unpublished)

Timeline (continued)

History

Hwang, Woo-Suk (Seoul National University)

1. Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, LeeBC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY.Evidence of a pluripotent human embryonic stem cell line derived from acloned blastocyst. Science 2004; 303:1669-74.

2. Hwang WS, Roh SI, Lee BC, Kang SK, Kwon DK, Kim S, Kim SJ, ParkSW, Kwon HS, Lee CK, Lee JB, Kim JM, Ahn C, Paek SH, Chang SS, KooJJ, Yoon HS, Hwang JH, Hwang YY, Park YS, Oh SK, Kim HS, Park JH,Moon SY, Schatten G. Patient-specific embryonic stem cells derived fromhuman SCNT blastocysts. Science 2005; 308:1777-83.

3. Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Shamim MH, Kim JJ,Kang SK, Schatten G, Hwang WS. Dogs cloned from adult somatic cells.Nature 2005; 436:641.

SCNT


totipotent

pluripotent

unipotent

pote

ncy

entire organism



hESC

mesoderm

endoderm

ectodermmultipotent

HSC


lymphocyte

neutrophil

monocyte

platelet

erythrocyte

History

cardiac muscle

panc acinus

neuron

History

Hwang, Woo-Suk (Seoul National University)

1. Hwang WS, Ryu YJ, Park JH, Park ES, Lee EG, Koo JM, Jeon HY, LeeBC, Kang SK, Kim SJ, Ahn C, Hwang JH, Park KY, Cibelli JB, Moon SY.Evidence of a pluripotent human embryonic stem cell line derived from acloned blastocyst. Science 2004; 303:1669-74.

2. Hwang WS, Roh SI, Lee BC, Kang SK, Kwon DK, Kim S, Kim SJ, ParkSW, Kwon HS, Lee CK, Lee JB, Kim JM, Ahn C, Paek SH, Chang SS, KooJJ, Yoon HS, Hwang JH, Hwang YY, Park YS, Oh SK, Kim HS, Park JH,Moon SY, Schatten G. Patient-specific embryonic stem cells derived fromhuman SCNT blastocysts. Science 2005; 308:1777-83.

3. Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, Shamim MH, Kim JJ,Kang SK, Schatten G, Hwang WS. Dogs cloned from adult somatic cells.Nature 2005; 436:641.

RETRACTED

RETRACTED

OK?

• Unethical process of acquiring human oocytes• All 11 of Hwang's stem cell lines were fabricated• Dismissed from SNU March 20, 2006

"I was blinded by work and my drive for achievement."

History

Omaha Experience1. Das AV, Mallya KB, Zhao X, Ahmad F, Bhattacharya S, Thoreson WB, Hegde GV, Ahmad I. Neural stem cell properties of

Muller glia in the mammalian retina: Regulation by Notch and Wnt signaling. Dev Biol 2006.2. Boer B, Bernadt CT, Desler M, Wilder PJ, Kopp JL, Rizzino A. Differential activity of the FGF-4 enhancer in F9 and P19

embryonal carcinoma cells. J Cell Physiol 2006; 208:97-108.3. Zhang JL, Cai J, Jackson JD, Kuszynski CA, Walls S, McIvor RS, Fox IJ. Long-term transgene expression and survival of

transgene-expressing grafts following lentivirus transduction of bone marrow side population cells. Transplantation 2005;79:882-8.

4. Peng H, Huang Y, Duan Z, Erdmann N, Xu D, Herek S, Zheng J. Cellular IAP1 regulates TRAIL-induced apoptosis in humanfetal cortical neural progenitor cells. J Neurosci Res 2005; 82:295-305.

5. Lee KM, Yasuda H, Hollingsworth MA, Ouellette MM. Notch 2-positive progenitors with the intrinsic ability to give rise topancreatic ductal cells. Lab Invest 2005; 85:1003-12

6. Hou J, Wilder PJ, Bernadt CT, Boer B, Neve RM, Rizzino A. Transcriptional regulation of the murine Elf3 gene in embryonalcarcinoma cells and their differentiated counterparts: requirement for a novel upstream regulatory region. Gene 2004;340:123-31.

7. Das AV, James J, Zhao X, Rahnenfuhrer J, Ahmad I. Identification of c-Kit receptor as a regulator of adult neural stem cellsin the mammalian eye: interactions with Notch signaling. Dev Biol 2004; 273:87-105.

8. Abe S, Boyer C, Liu X, Wen FQ, Kobayashi T, Fang Q, Wang X, Hashimoto M, Sharp JG, Rennard SI. Cells derived from thecirculation contribute to the repair of lung injury. Am J Respir Crit Care Med 2004; 170:1158-63.

9. Nagata H, Ito M, Cai J, Edge AS, Platt JL, Fox IJ. Treatment of cirrhosis and liver failure in rats by hepatocytexenotransplantation. Gastroenterology 2003; 124:422-31.

10. Lee KM, Nguyen C, Ulrich AB, Pour PM, Ouellette MM. Immortalization with telomerase of the Nestin-positive cells of thehuman pancreas. Biochem Biophys Res Commun 2003; 301:1038-44.

11. Beschorner WE, Sudan DL, Radio SJ, Yang T, Franco KL, Hill AC, Shearon CC, Thompson SC, Dixon RS, Johnson ND,Kuszynski CA, Rubocki RJ, Lechtenberg KF, Matamoros A, Jr., Goertzen TC, Fox IJ, Langnas AN. Heart xenograft survivalwith chimeric pig donors and modest immune suppression. Ann Surg 2003; 237:265-72.

12. Abe S, Lauby G, Boyer C, Rennard SI, Sharp JG. Transplanted BM and BM side population cells contribute progeny to thelung and liver in irradiated mice. Cytotherapy 2003; 5:523-33.

13. Zhao X, Das AV, Thoreson WB, James J, Wattnem TE, Rodriguez-Sierra J, Ahmad I. Adult corneal limbal epithelium: amodel for studying neural potential of non-neural stem cells/progenitors. Dev Biol 2002; 250:317-31.

14. Cai J, Ito M, Nagata H, Westerman KA, Lafleur D, Chowdhury JR, Leboulch P, Fox IJ. Treatment of liver failure in rats withend-stage cirrhosis by transplantation of immortalized hepatocytes. Hepatology 2002; 36:386-94.

Current Applications

Experimental/Proposed

1. Therapeutic Transplantation

• Myocardial infarction• Stroke• Alzheimer’s• Multiple sclerosis• Parkinson’s disease• Huntington’s disease• Spinal cord injury• Diabetes• Cartilage replacement• Etc., etc, etc.

2. Study of human development3. Drug/toxin testing4. Gene therapy(5. Reproductive cloning)

Established

1. Bone marrow transplantation

• Nonmodified• Genetically modified

2. In vitro fertilization (IVF)

“By promoting the falsehood that adult stem cell treatmentsare already in general use for 65 diseases and injuries,Prentice and those who repeat his claims mislead laypeopleand cruelly deceive patients.”

Neaves et al., Science 2006

Stem cells & wound healing


Some reports in minor journals of enhanced dermal and bone healingwith local or systemic administration of epidermal or mesenchymalstem cells (i.e., adult stem cells); no practical applications.


Potential problems with stem cell therapy

• Teratoma or other tumor

• Immune rejection/GVDH

• Dosage and delivery

• Targeting accuracy

• Exploitation

• Ethical conundrum


Hot treatment…or not

BMJ 2006;333:770Nature 2006;440:850

Conclusions

Stem Cell Therapy

1. Proofs of concept are readily available, but…

2. Translation into practical treatments have been rare

3. Road to practical treatments will be long and hard

“Tissue repair is designed to reconstruct morphological integrity;epimorphic regeneration is designed to restore function.”

RJ Goss, 1992

“When my liver fails, don't give me a bone marrow transplant,give me a liver.”

Irving Weissman, 2004

Take-home message:

Healing bad, regeneration good.

Documents

Stem Cell Therapy - Nebraska Surgical Researchnebraskasurgicalresearch.com/s NSR/presentation_pdf/2006... · 2004 Murine parthenogenesis (Kono, Tokyo) 2004-5 Hwang misconduct (Seoul),