STEM CELL ENGINEERING

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STEM CELL ENGINEERING

• Stem cells self-renew--divide to maintain
  themselves and produce progeny that will
  differentiate.
  
• Stem cells are pluripotent or multipotent-- they
  produce cells in many different lineages.
  
• Stem cells potency ranges from embryonic (most
  potent) to adult (more restricted).
  

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SELF-RENEWAL
MULTI-POTENCY
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Transfer of somatic nuclei into oocytes induces
complete reprogramming- work done at UMass
Amherst.
Cloned Transgenic Calves Produced from
Nonquiescent Fetal Fibroblasts
Jose B. Cibelli, Steve L. Stice, Paul J. Golueke,
Jeff J. Kane, Joseph Jerry, Cathy Blackwell, F.
Abel Ponce de León, James M. Robl
Science, Vol 280, Issue 5367, 1256-1258, 22 May
1998
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What is Nuclear Reprogramming?
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Efficiency of cloning is low.

• Less than 10 of the transferred Blastocysts
  survive to term. Further losses occur after
  birth. Derivation of embryonic stem cells is also
  highly inefficient.
• Therefore, it is important to design methods to
  improve reprogramming and other events that occur
  immediately after fertilization.

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Rafael Fissore, D.V.M, Ph.D.
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Rafael Fissore, D.V.M, Ph.D.
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Rafael Fissore, D.V.M, Ph.D.
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Rafael Fissore, D.V.M, Ph.D.
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ADULT HEMATOPOIETIC STEM CELLS
-bone marrow transplants for damaged immune
systems - in vitro differentiation to replace a
specific cell type
EXTREMELY RARE
DONT PROLIFERATE AND MAINTAIN
SELF-RENEWAL AND MULTI-POTENCY IN CULTURE
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Self-renewal LTR-HSC
Multi-potency
T cell
CD4 CD8- TCRbhigh
HSC
CD4 CD8 TCRbint
CD4- CD8- TCRb/-
CD34 KSL
CD34- KSL
CD8 CD4- TCRbhigh
STR-HSC
CLP
Sca-1- c-Kit Lin- CD34 FcgRII/III-
CD43 B220- AA4.1
CMP
CD43 B220int AA4.1
Sca-1- c-Kit Lin- CD34- FcgRII/III-
Sca-1- c-Kit Lin- CD34 FcgRII/III
CD43int B220int
GMP
MEP
B cell
CD43- B220high CD19 IgM
Meg.
RBC
Neu
Mo
Gr-1 CD11b
Gr-1- CD11b
Myeloid
Ter119
CD41
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Competitive reconstitution of bone marrow
chimeric mice
Assay mutant protein GFP cells
lethal
1200 rads
5-FU-treated BM cells infected with a retrovir
al vector encoding a mutant protein and green
fluorescent protein (GFP)

4 months Long term reconstitution HSC activi
ty
Increased percent mutant protein GFP expressing
cells in all lineages Expansion of HSC compar
tment

Janice Telfer, Ph.D.
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African trypanosomes
www.iaea.org/.../NAAL/ agri/ent/entTSETSEmain.php

• unicellular, protozoan parasites
• inhabit the extracellular compartments of host
  tissues
  
• cause sleeping sickness in humans and Nagana in
  cattle

http//tryps.rockefeller.edu/crosslab_intro.html
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Murine model of trypanosomiasis

• splenomegaly with a loss of normal splenic
  architecture
  
• loss of marginal zones

Infection and Immunity, Aug. 1994, p. 3230-5

• B cell proliferation and plasma cell
  differentiation
  
• hyperglobulinemia
• loss of the ability to make antibodies to
  specific antigens
  
• alterations of lymphoid populations

Immunobiology of African trypanosomes in
laboratory rodents, Ch5 http//www.fao.org/Wairdo
cs/ILRI/x5550E/x5550e09.htm
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Putative HSCs expand in the BM and spleen during
infection with T. brucei
Bone Marrow Stem cell analysis d7 post infection
uninfected

35
uninfected BM
30
GUTat 3.1 BM
25
d7
20
of FL-3 negative cells
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10
5
0
anti mouse-ckit-SA-APC (stem cell factor recept
or)
lin -
ckit/sca1
GUTat-infected
population
Splenic Stem cell analysis d7 post infection

35
d7
uninfected Spleen
30
GUTat 3.1 Spleen
25
20
of FL-3 negative cells
15

10
5
anti mouse-Sca1-PE
Lineage markers Gr-1(myeloid) Ter119 (erythrocyt
e)
CD19 (B cell) CD3 (T cell)
0
lin -
ckit/sca1
hematopoietic progenitor and stem cell subset.
population
7AAD
Samuel Black, Ph.D.
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• Induction of cell division in the egg- Rafael
  Fissore, Ph.D.
  
• Expansion of hematopoietic stem cells in culture-
  Janice Telfer, Ph.D.
  
• Expansion and mobilization of hematopoietic stem
  cells- Samuel Black, Ph.D.
  
• Three new faculty members in stem cells and
  tissue engineering