Stem Cell Plasticity Research for Regenerative Medicine

Jan A. Nolta, Ph.D.

Associate Professor, Division of Oncology/Hematopoietic Development and Malignancy Program

Washington University School of Medicine

Stem cell plasticity and cloning……..

• Why are these topics suddenly so prevalent?

• What are the key issues related to understanding stem cells and cloning?

Mature Tissues

Stem Cell

How are stem cells defined?

Differentiation and Commitment

1) Self-renewal

2) Multi-potential

3) Highly proliferative

Key Issues to Remember• There are many different types of stem cells.• An important distinction: Embryonic vs. Adult

1) Embryonic stem cells are derived from an egg shortly after the it begins to grow and are totipotent, able to generate any tissue of the body.

2) Adult stem cells are found in many different anatomical sites in the body and appear to have more restricted and specialized functions.

3) New research in the field of “Stem Cell Plasticity” has suggested that adult stem cells might have more regenerative potential than was previously suspected.

4) More research is needed to compare the regenerative potential of embryonic vs. adult stem cells.

Embryonic stem cells: Isolated from human tissue(1998)

Derivation of Embryonic Stem Cells

Totipotent ES Cells

The generation of federally approved human ES cell lines was all done in test tubes from eggs collected from human donors, salvaged from cryobanks.

The National Bioethics Advisory Commission recommended only using human ova left over from in

vitro fertilization procedures.

It was from these otherwise discarded cells that the 64 human ES cell lines were generated which were ultimately approved by the Bush Administration.

From these lines, only 8 are dividing continually and are available for use in the investigation into their potential

to be used in regenerative medicine (not currently studied at Wash U).

It is not known yet if they will be better, be able to repair more tissues, or live longer than stem cells from adult

sources. More comparisons are needed.

• Federally approved stem cell lines are derived from discarded eggs from fertility clinics.

The Cloning Process:Dolly the Sheep

Egg Cell(black-faced sheep)

Adult Mammary Cell(white-faced sheep)

Nucleus from the black faced sheep is removed and replaced with a nucleus from the white faced sheep

Nuclear transfer is done with a finely drawn glass pipette. Nuclei are shown in the pipette, ready to be injected into the recipient egg.

The Cloning Process:Dolly the Sheep

Surrogate Mother (black-facedSheep)

Egg Cell(black-faced sheep)

Adult Mammary Cell(white-faced sheep)

Dolly the sheep

Wilmut et al

Nature 1996 March7;380:64-6

Will human cloning be used to create new people?

Cloning human stem cells is beingStudied to learn how to regeneratea patient’s own damaged tissuesand organs, without the possibilityof rejection by the immune system.

NO!

Combining cloning with the use of embryonic stem cells

Egg Cell

Healthy Adult Cell: nuclear material “matches” the patients; no rejection

Generate humanEmbryonic stem cellswithout using sperm

Spinal cord injured patient

RegenerativeTherapy

Human Development

Stem cells also exist in many adult tissues

Embryonic Stem cells

Adult Stem Cells found in:Bone marrow

BrainLiver

PancreasSkin

MuscleIntestine

& other organs

Embryonic stem cells: Isolated from human eggs after in vitro fertilization OR donation of nuclear material from an already differentiated adult cell (cloning)

Characteristics of Adult Stem Cells

• Found in discreet anatomical sites in many major organ systems

• Typically, restricted in potential to the organ of residence (i.e. brain stem cells only make brain tissue, marrow stem cells make blood, etc.)

• In many cases, most readily evident as a reservoir of tissue for repair functions.

• Some studies have SUGGESTED that adult stem cells may be able to contribute to the repair of liver, brain, muscle, and other tissues.

Adult stem cells exist for many different human organs - can they serve as an alternative to embryonic stem cells?

Current use of adult stem cells:• Bone marrow transplantation

Potential uses of adult stem cells:• Brain and spinal cord injury and disease.• Repair of heart tissue.• Regeneration of liver tissue.• Diabetes therapy - pancreas repair.• Repair of muscle, blood vessels, and skin

(potential therapies for burn victims)• And many other possibilities……..

HSCBLOOD

VASCULAR STEM CELLS

HEPATIC STEM CELLS

MUSCLESTEM

CELLS

NEURAL STEM CELLSMESENCHYMAL

STEM CELLS

FAT

CARTILAGE

MUSCLE

CARDIACAND

SKELETAL

BONE

FIBRO-BLAST

NEURONS

WHOLEMOUSE

LIVER

?

?

Multipotent Adult Progenitor Cells (MAPC) can be generated from multiple tissues

Brain

Muscle

Marrow

vWF GFAPNF-200

HNF-1Albumin

1E+2

1E+4

1E+6

1E+8

1E+10

1E+12

1E+14

1E+16

1E+18

1E+20

1E+22

0 50 100 150 200 250

BMMuscle Brain

Fold

in

cre

ase

Reyes, Verfaillie et al, “Multipotent Adult Progenitor Cells”

Nature 2002 Endothelium, neuron, liver

Human Mesenchymal Stem Cells fromBone Marrow and Adipose Tissue

Todd E. Meyerrose, Phillip Herrbrich, David A. Hess, Todd E. Meyerrose, Phillip Herrbrich, David A. Hess, Gordon D. Wu, and Jan A. NoltaGordon D. Wu, and Jan A. Nolta

Washington University School of MedicineWashington University School of MedicineSt. Louis, MissouriSt. Louis, Missouri

www.jannoltalab.comwww.jannoltalab.com

MESENCHYMAL STEM CELLS

FAT

CARTILAGE

MUSCLE

CARDIAC AND

SKELETAL

BONE

FIBRO-BLAST

NEURONS??

??

Phenotype of the most primitive MSC populations?Phenotype of the most primitive MSC populations?

Trafficking properties in vivo? In response to injury?Trafficking properties in vivo? In response to injury?

Bone Bone marrowmarrow

Adipose Adipose TissueTissue

Adipose-derived mesenchymal stem

cell (AMSC)

Fat

Bone Cartilage

Muscle

Generation of Adipose-derived MSC (AMSC)

Isolation procedure:

• Take a bucket to the OR; collect fat excised during liposuction (LA) or gastric bypass (St Louis)

• Dissect away visible vessels and mince• Collagenase digest and separate by density centrifugation• +/- HSC removal• Defined initially by plastic adherence and rapid growth in

minimal mediuum• Easily transduced and very proliferative

• Collaborators: De Ugarte and Hedrick: UCLAChris Eagon, Wash U. St Louis

AMSC numbers increase rapidly after plating

Zuk et al, Tissue engineering 2001

Day 165

2 x 10 10

Cells fromOne flaskinitiallyseeded

Neo-organoid (new tissue) created from adipose – derived adult stem cells seeded

onto a biodegradable matrix

• The tissue was created by implanting human AMSC on the matrix into a laboratory strain of mice that have no immune system. It is the size of a dime.

• Matrix is very well tolerated, non-immunogenic, and permissive for neo-vascularization and systemic protein delivery.

• We are currently using these organoids to deliver therapeutic drugs and proteins such as clotting factors needed in hemophiliacs, and we are studying their potential to form specific tissues such as a new pancreas for diabetic patients.

Nude/NOD/SCIDMouse Tissue

INT HRT SPL LIV LUNG KID MUS BRAIN FAT

Positive for human AMSC (30-75 days)

11 7 13 13 14 11 12 7 10

Total tested by PCR 14 13 16 16 16 16 16 14 12

**

*Other MSC types (human BM or UCB-derived MSC have not been *Other MSC types (human BM or UCB-derived MSC have not been detected migrating into the brains of highly perfused immune deficient micedetected migrating into the brains of highly perfused immune deficient mice

AMSC trafficking in immune deficient miceAMSC trafficking in immune deficient mice

kidney lung

spleen liver

This data shows that injected adipose and bone This data shows that injected adipose and bone marrow –derived MSC lodge in multiple tissues marrow –derived MSC lodge in multiple tissues following various routes of administration into following various routes of administration into sublethally irradiated immune deficient mice sublethally irradiated immune deficient mice ((Meyerrose et al, Ms in prepMeyerrose et al, Ms in prep))

Yet in models of acute local injury, MSC appear Yet in models of acute local injury, MSC appear to preferentially home to, or accumulate in, the to preferentially home to, or accumulate in, the damaged tissue (damaged tissue (Wu, Nolta et al, Transplantation Wu, Nolta et al, Transplantation 20032003))

Robust migration of β-Gal+ mesenchymal cells into heart allografts during the development of chronic rejection. (A) lacz-Labeled mesenchymal cells (blue) at inflammatory zone close to subendocardial area. (B) Tissues in the outer layer of allograft. (C) Perivascular area D) Mature fibrosis lesion. (Wu, Nolta et al 2003)

Intravenous engraftment of Mesenchymal stem cells into rat recipients of heart transplant resulted in preferential migration of the infused MSC into areas of injury in the cardiac grafts.

Mechanisms for recruitment of adult stem cells to sites of damage are currently under investigation.

Migration of mesenchymal stem cells to heart allografts during chronic rejection. Wu GD, Nolta JA, Jin YS, Starnes VA, Cramer DV. Transplantation. 2003; 75:679-85.

Bone Regeneration: Mesenchymal stem cells (MSC)Bone Regeneration: Mesenchymal stem cells (MSC)

MSCs repair large gaps in bones in 32 weeks

No evidence of immune rejection following implantation

of MSC from an unrelated donor

MSCs repair large gaps in bones in 32 weeks

No evidence of immune rejection following implantation

of MSC from an unrelated donor

Phase 2 clinical trials for large cranio-facial and long bone defects began in 2002

in Europe and US

Phase 2 clinical trials for large cranio-facial and long bone defects began in 2002

in Europe and US

ControlControl MSC treatedMSC treated

MSCs protect cartilage and boneMSCs protect cartilage and bone

Phase 1 clinical trial began in 2002 in Europe and US

Phase 1 clinical trial began in 2002 in Europe and US

Meniscal Repair - Cartilage ProtectionMeniscal Repair - Cartilage Protection

Complete menisectomyComplete menisectomy Partial menisectomyPartial menisectomy

goat model

Summary: MSC - Uses

Cell therapy for genetic disease:

Relatively robust systemic protein delivery

Tissue regeneration: bone, cartilage, skeletal (and cardiac) muscle,

supportive tissue in spine and brain

Basic stem cell biology; study recruitment to damaged tissues and the

mechanisms involved in trans-differentiation

HSCBLOOD

HEPATIC STEM CELLS (oval cells??)

LIVER ??

Liver “plasticity” project – Nolta LabLiver “plasticity” project – Nolta Lab

Human Albumin+ Hepatocyte-like cells were generated from human UCB CD34+/CD38-/CD7- cells in

immunodeficient mouse liver, following liver injury by CCl4 and administration of human Hepatocyte Growth Factor (HGF).

Human Albumin+Cells in Mouse liver

Human Albumin - negativeCells in Mouse liver: Likely hematopoietic

In situ hybridization for human ALU sequences coupled with In situ hybridization for human ALU sequences coupled with FITC-labeled anti-human albumin antibodyFITC-labeled anti-human albumin antibody

HumanHumanMurineMurine

In situ hybridization for human ALU sequencesIn situ hybridization for human ALU sequences

CC

l4

CC

l4+

HG

F

Hep

G2

BM

Spl

een

No

CC

l4

Hepatocytes

CD

34+

CD

38-C

D7-

CB

MN

C

CD

34+

Starting HSC population

Non-transplanted murine hepatocytes

CC

l4

No

Tx

Other tissues

Human-mouse chimeric

M 2M H2M

Albumin

MW

Liver “Plasticity” Project Summary

• Human umbilical cord blood - derived CD34+ or CD34+CD38-CD7- cells can differentiate into albumin - expressing cells in the damaged livers of immunodeficient mice.

• Massive liver damage was necessary to induce albumin expression from hematopoietic cells that seed to the liver. Administration of HGF increases the levels of albumin expression.

Albumin-expressing hepatocyte-like cells develop in the livers of immune-Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice transplanted with highly purified human stem cells. deficient mice transplanted with highly purified human stem cells.

Wang X, Ge S, McNamara G, Hao QL, Crooks GM, and Nolta JA.Wang X, Ge S, McNamara G, Hao QL, Crooks GM, and Nolta JA. Blood, 101: 4201-4208, 2003 Blood, 101: 4201-4208, 2003

Bone Marrow-Derived Stem Cells Initiate

Pancreatic Regeneration

Hess David A., Li L, Martin M, Sakano S, Hill

D, Strutt B, Thyssen S, Gray DA, Bhatia M

Nature Biotechnology 2003 Jul;21(7):

763-70.

Conclusions-Stem Cell Mediated Tissue Repair

• There are stem cells in many areas of the human body other than bone marrow: including fat

– These alternative sources may be useful for different applications in tissue engineering and cell-based therapy

• Injury models, both chronic and acute, are needed to promote more robust engraftment, and this may be a physiologic process that is ongoing in injury until the potential for repair is exceeded.

• Immune deficient mice allow the study of human stem cell recruitment and differentiation during tissue repair and remodeling. Other animal models are also moving the research forward significantly.

Summary: Stem Cells and Cloning

• Stem cells can be derived from both embryonic and adult tissues.

• In principle, cloning procedures can be used to generate embryonic stem cells from any source.

• Embryonic and adult stem cells have enormous potential to advance medical technology, and more research is needed for both types of cell.

Current Students/LabTodd Meyerrose (grad student)

Dave Hess (Post-Doctoral Fellow)Phillip Herrbrich (tech II)

Ivana Rosova (grad student)Timothy Craft (tech)Louisa Wirthlin (tech)Laura Eichholz (tech)

Gerhard Bauer (GMP Facility Director: Wash U)Jonny Walker (QA/SOPs: Wash U GMP facility)

Mo DaoJesusa Arevalo

Xiuli Wang Janice Arakawa-Hoyt

Eleanor TsarkShundi Ge

Joseph HwaDelia Ertl Isabel KuoKenny Xi

Gordon Wu

Former Students

Thank you…………….

Collaborators

Mark SandsAlex Hofling

Steve DevineJohn Dipersio

Dan LinkKathy Ponder

Stem Cell Information Sources

National Institutes of HealthStem Cell Information Page (broad comprehensive information)http://www.nih.gov/news/stemcell/index.htm

American Association for the Advancement of Science (AAAS)Stem Cell Informtion (Research, Ethical, and Policy issues)http://www.aaas.org

National Academy of SciencesStem Cells and the Future of Regenerative Medicine (Full text

online):http://www.nap.edu/books/0309076307/html

University of Wisconsin-MadisonStem Cell Research Home Page (Research and technology):http://www.news.wisc.edu/packages/stemcells/index.html