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Stem Cell Plasticity Research
for Regenerative Medicine
Jan A. Nolta, Ph.D.
Associate Professor, Division of Oncology/
Hem...
Stem cell plasticity and cloning……..
• Why are these topics suddenly so
prevalent?
• What are the key issues related to
un...
Mature
Tissues
Stem Cell
How are stem cells
defined?
Differentiation and Commitment
1) Self-renewal
2) Multi-potential
3) ...
Key Issues to Remember
• There are many different types of stem cells.
• An important distinction: Embryonic vs. Adult
1) ...
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 do...
The National Bioethics Advisory Commission
recommended only using human ova left over from in
vitro fertilization procedur...
• 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 ...
Nuclear transfer is done with a finely drawn glass pipette. Nuclei are
shown in the pipette, ready to be injected into the...
The Cloning Process:
Dolly the Sheep
Surrogate
Mother (black-faced
Sheep)
Egg Cell
(black-faced sheep)
Adult Mammary Cell
...
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 being
Studied to learn how to regenerate
a pa...
Combining cloning with the
use of embryonic stem cells
Egg Cell
Healthy Adult Cell: nuclear material
“matches” the patient...
Human Development
Stem cells also exist in many adult tissues
Embryonic Stem cells
Adult Stem Cells found in:
Bone marrow
Brain
Liver
Pancreas
Skin
Muscle
Intestine
&
other organs
Embryonic stem
cells: Iso...
Characteristics of Adult Stem Cells
• Found in discreet anatomical sites in many
major organ systems
• Typically, restrict...
Adult stem cells exist for many different
human organs - can they serve as an
alternative to embryonic stem cells?
Current...
HSCBLOOD
VASCULAR STEM CELLS
HEPATIC STEM CELLS
MUSCLE
STEM
CELLS
NEURAL STEM CELLS
MESENCHYMAL
STEM CELLS
FAT
CARTILAGE
M...
Multipotent Adult Progenitor Cells (MAPC) can be
generated from multiple tissues
Brain
Muscle
Marrow
vWF GFAP
NF-200
HNF-1...
Human Mesenchymal Stem Cells from
Bone Marrow and Adipose Tissue
Todd E. Meyerrose, Phillip Herrbrich, David A. Hess,Todd ...
MESENCHYMAL
STEM CELLS
FAT
CARTILAGE
MUSCLE
CARDIAC
AND
SKELETAL
BONE
FIBRO-
BLAST
NEURONS
??
??
Phenotype of the most pri...
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
liposu...
AMSC numbers increase rapidly after plating
Zuk et al, Tissue engineering 2001
Day 165
2 x 10 10
Cells from
One flask
init...
Neo-organoid (new tissue) created from
adipose – derived adult stem cells seeded
onto a biodegradable matrix
• The tissue ...
Nude/NOD/SCID
Mouse Tissue
INT HRT SPL LIV LUNG KID MUS BRAIN FAT
Positive for human
AMSC (30-75 days)
11 7 13 13 14 11 12...
kidney lung
spleen liver
This data shows that injected adipose and boneThis data shows that injected adipose and bone
marrow –derived MSC lodge in ...
Robust migration of β-Gal+ mesenchymal cells into heart allografts during the
development of chronic rejection. (A) lacz-L...
Intravenous engraftment of Mesenchymal stem cells
into rat recipients of heart transplant resulted in
preferential migrati...
Bone Regeneration: Mesenchymal stem cells (MSC)
MSCs repair large gaps in
bones in 32 weeks
No evidence of immune
reject...
Control MSC treated
MSCs protect cartilage and bone
Phase 1 clinical trial
began in 2002 in
Europe and US
Phase 1 clinical...
Summary: MSC - Uses
Cell therapy for genetic disease:
Relatively robust systemic protein delivery
Tissue regeneration: bon...
HSCBLOOD
HEPATIC STEM CELLS (oval cells??)
LIVER ??
Liver “plasticity” project – Nolta LabLiver “plasticity” project – Nol...
Human Albumin+ Hepatocyte-like cells were generated
from human UCB CD34+/CD38-/CD7- cells in
immunodeficient mouse liver, ...
Human Albumin+
Cells in Mouse liver
Human Albumin - negative
Cells in Mouse liver:
Likely hematopoietic
In situ hybridizat...
HumanHumanMurineMurine
In situ hybridization for human ALU sequencesIn situ hybridization for human ALU sequences
CCl4
CCl4+HGF
HepG2
BM
Spleen
NoCCl4
Hepatocytes
CD34+CD38-CD7-
CBMNC
CD34+
Starting HSC
population
Non-transplanted
murin...
Liver “Plasticity” Project Summary
• Human umbilical cord blood - derived CD34+ or
CD34+CD38-CD7- cells can differentiate ...
Bone Marrow-Derived
Stem Cells Initiate
Pancreatic
Regeneration
Hess David A., Li L,
Martin M, Sakano S, Hill
D, Strutt B,...
Conclusions-Stem Cell Mediated
Tissue Repair
• There are stem cells in many areas of the human body
other than bone marrow...
Summary: Stem Cells and Cloning
• Stem cells can be derived from both
embryonic and adult tissues.
• In principle, cloning...
Current Students/Lab
Todd Meyerrose (grad student)
Dave Hess (Post-Doctoral Fellow)
Phillip Herrbrich (tech II)
Ivana Roso...
Stem Cell Information Sources
National Institutes of Health
Stem Cell Information Page (broad comprehensive information)
h...
Slide 1 - Washington University in St. Louis
Slide 1 - Washington University in St. Louis
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Slide 1 - Washington University in St. Louis

  1. 1. 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
  2. 2. Stem cell plasticity and cloning…….. • Why are these topics suddenly so prevalent? • What are the key issues related to understanding stem cells and cloning?
  3. 3. Mature Tissues Stem Cell How are stem cells defined? Differentiation and Commitment 1) Self-renewal 2) Multi-potential 3) Highly proliferative
  4. 4. 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.
  5. 5. Embryonic stem cells: Isolated from human tissue (1998)
  6. 6. 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.
  7. 7. 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.
  8. 8. • Federally approved stem cell lines are derived from discarded eggs from fertility clinics.
  9. 9. 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
  10. 10. Nuclear transfer is done with a finely drawn glass pipette. Nuclei are shown in the pipette, ready to be injected into the recipient egg.
  11. 11. The Cloning Process: Dolly the Sheep Surrogate Mother (black-faced Sheep) Egg Cell (black-faced sheep) Adult Mammary Cell (white-faced sheep)
  12. 12. Dolly the sheep Wilmut et al Nature 1996 March7; 380:64-6
  13. 13. Will human cloning be used to create new people? Cloning human stem cells is being Studied to learn how to regenerate a patient’s own damaged tissues and organs, without the possibility of rejection by the immune system. NO!
  14. 14. Combining cloning with the use of embryonic stem cells Egg Cell Healthy Adult Cell: nuclear material “matches” the patients; no rejection Generate human Embryonic stem cells without using sperm Spinal cord injured patient Regenerative Therapy
  15. 15. Human Development Stem cells also exist in many adult tissues Embryonic Stem cells
  16. 16. Adult Stem Cells found in: Bone marrow Brain Liver Pancreas Skin Muscle Intestine & 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)
  17. 17. 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
  18. 18. 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……..
  19. 19. HSCBLOOD VASCULAR STEM CELLS HEPATIC STEM CELLS MUSCLE STEM CELLS NEURAL STEM CELLS MESENCHYMAL STEM CELLS FAT CARTILAGE MUSCLE CARDIAC AND SKELETAL BONE FIBRO- BLAST NEURONS WHOLE MOUSE LIVER ? ?
  20. 20. Multipotent Adult Progenitor Cells (MAPC) can be generated from multiple tissues Brain Muscle Marrow vWF GFAP NF-200 HNF-1 Albumin 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 BM Muscle Brain Foldincrease Reyes, Verfaillie et al, “Multipotent Adult Progenitor Cells” Nature 2002 Endothelium, neuron, liver
  21. 21. Human Mesenchymal Stem Cells from Bone 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 Medicine St. Louis, MissouriSt. Louis, Missouri www.jannoltalab.comwww.jannoltalab.com
  22. 22. 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? BoneBone marrowmarrow AdiposeAdipose TissueTissue
  23. 23. Adipose-derived mesenchymal stem cell (AMSC) Fat Bone Cartilage Muscle
  24. 24. 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: UCLA Chris Eagon, Wash U. St Louis
  25. 25. AMSC numbers increase rapidly after plating Zuk et al, Tissue engineering 2001 Day 165 2 x 10 10 Cells from One flask initially seeded
  26. 26. 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.
  27. 27. Nude/NOD/SCID Mouse 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
  28. 28. kidney lung spleen liver
  29. 29. This data shows that injected adipose and boneThis data shows that injected adipose and bone marrow –derived MSC lodge in multiple tissuesmarrow –derived MSC lodge in multiple tissues following various routes of administration intofollowing various routes of administration into sublethally irradiated immune deficient micesublethally irradiated immune deficient mice ((Meyerrose et al, Ms in prepMeyerrose et al, Ms in prep)) Yet in models of acute local injury, MSC appearYet in models of acute local injury, MSC appear to preferentially home to, or accumulate in, theto preferentially home to, or accumulate in, the damaged tissue (damaged tissue (Wu, Nolta et al, TransplantationWu, Nolta et al, Transplantation 20032003))
  30. 30. 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)
  31. 31. 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.
  32. 32. 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 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
  33. 33. Control MSC treated MSCs 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 Protection Complete menisectomy Partial menisectomy goat model
  34. 34. 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
  35. 35. HSCBLOOD HEPATIC STEM CELLS (oval cells??) LIVER ?? Liver “plasticity” project – Nolta LabLiver “plasticity” project – Nolta Lab
  36. 36. 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).
  37. 37. Human Albumin+ Cells in Mouse liver Human Albumin - negative Cells in Mouse liver: Likely hematopoietic In situ hybridization for human ALU sequences coupled withIn situ hybridization for human ALU sequences coupled with FITC-labeled anti-human albumin antibodyFITC-labeled anti-human albumin antibody
  38. 38. HumanHumanMurineMurine In situ hybridization for human ALU sequencesIn situ hybridization for human ALU sequences
  39. 39. CCl4 CCl4+HGF HepG2 BM Spleen NoCCl4 Hepatocytes CD34+CD38-CD7- CBMNC CD34+ Starting HSC population Non-transplanted murine hepatocytes CCl4 NoTx Other tissues Human-mouse chimeric M β2M Hβ2M Albumin MW
  40. 40. 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, 2003Blood, 101: 4201-4208, 2003
  41. 41. 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.
  42. 42. 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.
  43. 43. 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.
  44. 44. Current Students/Lab Todd 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 Dao Jesusa Arevalo Xiuli Wang Janice Arakawa-Hoyt Eleanor Tsark Shundi Ge Joseph Hwa Delia Ertl Isabel Kuo Kenny Xi Gordon Wu Former Students Thank you……………. Collaborators Mark Sands Alex Hofling Steve Devine John Dipersio Dan Link Kathy Ponder
  45. 45. Stem Cell Information Sources National Institutes of Health Stem 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 Sciences Stem Cells and the Future of Regenerative Medicine (Full text online): http://www.nap.edu/books/0309076307/html University of Wisconsin-Madison Stem Cell Research Home Page (Research and technology): http://www.news.wisc.edu/packages/stemcells/index.html

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