Stem Cells Lisheng WANG Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa

Aim and Expectation  Understand the important concepts  Gain the basic knowledge of stem cells  Know the frontier and controversial questions in the stem cell research field  Capable of reading and understanding stem cell literature  Capable of evaluating stem cell literature

Materials and methods used in the class  Slides (with references)  Stem Cell Glossary  Lectures  Short class discussion  Quiz after lecture  Literature for evaluation

How to learn  Show up  Concentrate on the slides labeled with square orange color (left upper corner, important slides!)  Complete quiz after class and discuss them with your classmates, which will help you to understand the important contents (also good for final exam).  Stop me anytime if you do not understand or you cannot hear me.  (If you are very interested in certain topics, please read reference paper, or search PubMed and/or Google Scholar using the keywords and author name provided in the slides)

Stem cells  Lecture 1: Introduction of stem cells (Part I)  Lecture 2: Introduction of stem cells (Part II)  Lecture 3: Surrogate markers to characterize adult stem cells  Lecture 4: Adult stem cells - HSC  Lecture 5: Adult stem cells & applications  Lecture 6: Human pluripotent stem cells  Lecture 7: Epigenetic reprogramming (Part I)  Lecture 8: Epigenetic reprogramming (Part II)  Lecture 9: Differentiation of pluripotent stem cells & class summary

Lecture 1: Introduction of Stem Cells (Part I)  What are stem cells  Types of stem cells  Some general concepts of stem cells  Application of stem cells

What are stem cells? Renew themselves for long periods through cell division (self-renewal) Give rise to specialized cell types (differentiation) ← indicating an important slide

Types of Stem cells (Defined by their origin and potentials) Adult (or somatic) stem cells  Tissue specific adult stem cells  Mesenchymal stem cells Pluripotent stem cells  Embryonic stem cells (ES)  Embryonic germ cells (EG)  Embryonic carcinoma cells  Epiblast-derived stem cells  Induced pluripotent stem cells (iPS)

Type of Stem Cells Somatic Stem Cell  derived from developing or adult organism  restricted replicative potential in vitro  multipotent Embryonic Stem Cell  derived from blastocyst or primordial germ cells (PGC)  dividing without limit  pluripotent (capacity to make all 3 germ layers)

Developmental potentials  A multipotent cell can give rise to several types of mature cells  A pluripotent cell can give rise to almost all types of adult cells  A totipotent cell can give rise to a new individual, given appropriate maternal support

Developmental potentials Hematopoietic stem cells Multipotent

Adult (Somatic) stem cells 1.Emerge later in the fetus development 2.Serve as a reservoir of continuous replenishment for the tissues in which they reside

Nature 441, (29 June 2006) Adult stem cells: serve as a reservoir of continuous replenishment for the tissues in which they reside (homeostasis and repair) & with tissue heterogeneity

An adult stem cell-derived cell lineage (mouse) Handbook of Stem Cells

Mesenchymal stem/stromal cells Originally, MSCs (a heterogeneous population) represent the cells of non- hematopoietic (blood forming) stromal origin, residing in the bone marrow as blood stem cell niche. Currently, MSCs have been isolated from almost all other tissues. For instance: peripheral blood; umbilical cord blood; synovial membrane; deciduos teeth; amniotic fluid … (Abdallah, BM. Gene Therapy 2008; 15:109–116) Stem Cells Aug;28(8): , TRENDS in Immunology 2007; 28:

Mesenchymal stem/stromal cells Dominici M, et al, Cytotherapy 2006;8:315 – 317. Stem Cells Aug;28(8): The MSC is defined according to three criteria (according to the International Society for Cellular Therapy (ISCT) proposal): (more potentials?) (1) its property of adherence to plastic (2) its phenotype: CD14- or CD11b-, CD19- or CD79  -, CD34-, CD45-, HLA-DR-, CD73+, CD90+, CD105+ (3) its capacity to be differentiated into three lineages: chondrocyte, osteoblast and adipocyte (possible more).

Multipotent adult stem cells (MAPCs)?? Verfaillie CM. Novartis Found Symp. 2005;265:55-61 Raff, M. Annu. Rev. Cell Dev. Biol. 19, 1–22 (2003). Check E. Nature Mar 29;446(7135): Bone marrow cells can give rise to multipotent adult progenitor cells (MAPCs) after in vitro culture (Jiang et al., 2002). However, they are difficult to be cultured and the data are not readily to be reproduced. Lessons learned from stem cell research (controversial )

Multipotent adult spermatogonial-derived stem cells (MASCs, mouse) Marco Seande et al. Nature 2007; 449: 346 Stem Cells Jan;27(1):  Adult spermatogonial progenitor cells (SPCs) can give rise to multipotent adult spermatogonial-derived stem cells (MASCs).  MASCs can generate derivatives/cells (for instance, blood vessel cells, cardiac tissues) of three germ layers (i.e. ectoderm, mesoderm, endoderm) – (multipotent? Or pluripotent?).

Important questions about adult stem cells How many kinds of adult stem cells exist, and in which tissues do they exist? What are the sources of adult stem cells in the body - "leftover" embryonic stem cells, or arise in some other way? Why do they remain in an undifferentiated state when all the cells around them have differentiated? Do adult stem cells normally exhibit transdifferentiation (plasticity)? Is it possible to expand sufficient adult stem cells in vitro for transplants? Which factors stimulate stem cells to relocate to the sites of injury or damage? How to reduce immune rejection (allograft) Open questions

Types of Stem cells (Defined by their origin and potentials) Adult (or somatic) stem cells  Tissue specific adult stem cells  Mesenchymal stem cells Pluripotent stem cells  Embryonic stem cells (ES)  Embryonic germ cells (EG)  Embryonic carcinoma cells  Epiblast-derived stem cells (EpiSC)  Induced pluripotent stem cells (iPS)

Embryonic germ cells & embryonic carcinoma cells

Epiblast derived stem cells (EpiSCs, mouse) Nature Jul 12;448(7150):191-5; Nature Jul 12;448(7150): Mouse embryonic stem cells Mouse epiblast derived stem cells

Epiblast derived stem cells (EpiSCs, mouse)  Derived from the epiblast of postimplantation mouse embryos, a later developmental stage than the blastocysts (5.75 days post coitum (d.p.c.)/embryonic day (E)5.75).  Self-renewal and pluripotency  Distinct from mESCs in gene expression patterns while consistent with the characteristics of hESCs. Nature Jul 12;448(7150):191-5; Nature Jul 12;448(7150):196-9.

Human embryonic stem cells 1.Derived from a 5-6 days of blastocyte 2.In vitro develop into three germ layers and give rise to almost any type of cell - similar to in vivo inner cell mass development. If injected into immune deficient mouse, will form teratomas. 3.Show a spatial (location) and temporal (timing) developmental pattern

1. An immortal embryonic stem cell line is derived from a ~5-day blastocyte (Clive Cookson, Sci Am Jul;293(1):A6-11)

2. Similar to in vivo inner cell mass development, embryonic stem cells can in vitro develop into three germ layers after cellular aggregation (embryoid body), giving rise almost any type of tissue. ES cellsEmbryoid body (Clive Cookson, Sci Am Jul;293(1):A6-11)

15d-EB, 14d-CFUx100, Hoffman 21-day EB x200 Example: human embryoid body  Mesoderm  cardiac muscles

3. Show spatial and temporal development pattern, may recapitulate in vivo embryo development

Some important questions about human embryonic stem cells  What are the mechanisms that allow hESCs to proliferate in vitro without differentiation?  Are the hESCs heterogeneous and/or "partially" differentiated?  What are the cellular and molecular signals that are important in activating a human pluripotent stem cell to begin differentiating into a specialized cell type?  What stage of differentited stem/progenitor cells from hESCs will be best for transplantation?  What are potential clinical complications using hESC- derivatives and how to avoid teratoma  How will knowledge of the mechanisms that underlie self-renewal and differentiation help us to develop patient-specific therapies?

The differences between embryonic and adult stem cells 1.Source of origin 2.Proliferation capacity: ES >>> adult stem cells in cell cutlure  number cells for transplant 3.Differentiation capacity: ES >> adult stem cells in cell cutlure 4.Pluripotent capacity: ES but not adult stem cells  almost any type of cell 5.In vivo teratoma formation: undifferentiated ES but not adult stem cells  safety issue

Review Stem Cell Glossary