Stemcell

What is a stem cell?

Why is it important to know?

Mervin C. Yoder, MD Richard and Pauline Klingler Professor of Pediatrics and of Biochemistry and Molecular Biology and of Cellular and Integrative Physiology Indiana University School of Medicine Indianapolis, IN 1

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Mead Johnson sponsors programs such as this to give healthcare professionals access to scientific and educational information provided by experts. Dr. Yoder has complete and independent control over the planning and content of his presentation, and he is not receiving any compensation from Mead Johnson for this presentation. Dr. Yoder’s comments and opinions are not necessarily those of Mead Johnson. In the event that his presentation contains statements about uses of drugs that are not within the drugs' approved indications, Mead Johnson does not promote the use of any drug for indications outside the FDA-approved product label. 3

What is a stem cell?

Why is it important to know?

Mervin C. Yoder, MD Richard and Pauline Klingler Professor of Pediatrics and of Biochemistry and Molecular Biology and of Cellular and Integrative Physiology Indiana University School of Medicine Indianapolis, IN 4

Objectives 1. Define stem cells 2. Discuss derivation of embryonic stem cells and associated ethical questions 3. Discuss “therapeutic cloning” and the associated ethical questions 4. Discuss derivation of adult stem cells and discuss some limitations to use of these cells in the clinic

Many tissues of the fully developed organism require a mechanism be present for the replacement of aged, injured or diseased cells.

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Many tissues of the fully developed organism require a mechanism be present for the replacement of aged, injured or diseased cells. Turnover of blood cells in human circulation = 108-109 cells per hour Epithelial turnover in the small intestine = every 3-5 days Skin cell turnover is location dependent but = every 3-4 weeks on average 7

Stem cell definition Cells that reside in specific locations (niches), that are not fully differentiated (displaying functions typical of mature cells of that tissue), and possess controlled but robust proliferative potential (at least for the lifetime of the host).

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Stem cell definition Stem cells display the ability to self-renew (to divide and give rise to other stem cells) and to differentiate along specific molecular pathways to form cells of specialized functions.

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Stem cell definition Cells that possess the individual potential (clonality) to regenerate the stem cells and the differentiated progeny required to reconstitute the tissue lineage of origin;

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Stem cell definition Cells that possess the individual potential (clonality) to regenerate the stem cells and the differentiated progeny required to reconstitute the tissue lineage of origin; For example: A hematopoietic stem cell must make more hematopoietic stem cells and all the different kinds of blood cells required for normal physiology when transplanted into the host 11

Stem cell definition Stem cells display the ability to selfrenew (to divide and give rise to other stem cells) and to differentiate along specific molecular pathways to form cells of specialized functions. Self-renewal division = stem cell maintenance Differentiation division = daughter cell production 12

Stem cell definition Stem cells display the ability to selfrenew (to divide and give rise to other stem cells) and to differentiate along specific molecular pathways to form cells of specialized functions. Differentiation division = daughter cell production Differentiation division = daughter cell production 13

Stem cell definition Stem cells display the ability to selfrenew (to divide and give rise to other stem cells) and to differentiate along specific molecular pathways to form cells of specialized functions. Self-renewal division

+ Self-renewal division

= 14

Stem cell expansion

Definitions Two major classes of stem cells • Embryonic stem cells (ES) are derived from cells of mammalian preimplantation embryos (mouse, chick, pig, rhesus, bovine, human). • Adult (somatic; postnatal) stem cells represent the self-renewing cells in skin, hair, intestine, liver and hematopoietic tissues of fetal and adult subjects

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Depiction of a human zygote

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Two-cell stage

Eight-cell stage

Four-cell stage

Sixteen-cell stage 17

inner cell mass cells

(trophectoderm)

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Murine embryonic stem (ES) cell isolation M. Evans and M. Kaufmann, Nature 292:154, 1981 G. Martin, Proc Natl Acad Sci USA 78:7634, 1981

Cells from the inner cell mass of murine blastocysts can be grown in cultures in the presence of murine embryonic fibroblasts and will form stable cell lines that retain pluripotent properties. The cell lines that emerge are called embryonic stem (ES) cells.

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Murine ES cells growing on embryonic fibroblasts

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BLASTOCYST

Pluripotent stem cell

inner cell mass

Totipotent stem cell

culture

ZYGOTE

Embryonic stem cell (ES-cell) Pluripotent somatic stem cell

CNS

PNS

Hema.

Liver

Skin

Mesen.

etc.

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Derivation of human embryonic stem (ES) cells JA Thomson et al. Science 282, 145, 1998

Human blastocyst-derived pluripotent cell lines with normal karyotypes can be propagated for more than four months with retention of pluripotency.

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What is so controversial about the use of ES cells in research? From an ethical perspective, it is the derivation of ES cells - which requires the death of an embryo - that is problematic, not the use of ES cells in research.

To what extent does the human embryo have moral status? Consensus on the moral status of embryonic life has proven elusive, if not impossible, and it seems that the definitions are so greatly differing as to defy compromise. IUCB SCSG 2002 At least three common views are quoted: Full moral status No moral status Developmental moral status

“Full” moral status of embryos • Status is equivalent to that of an adult – Should we as a society provide the same degree of protection to the developing embryo that we ascribe for infants and small children? – Would we impose criminal penalties upon mothers who violate policies known to harm the embryo? 27

No moral status of embryos • Status is that of any other cluster of human cells • Is the embryo just a tissue (or subject)?

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Developmental moral status of the embryo The embryos deserves “respect” as a form of human life. The moral status of the embryo increases, and therefore the respect accorded to it increases, as it matures and develops. National Bioethics Advisory Comm. 1999 What does “respect” entail (given that the embryo is destroyed in the act of deriving the ES cells)?

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Potential Major Advance Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres Chung Y, et al. Nature 2006; 439:216-219

“We report an alternative method of establishing mES cell lines, using a technique of single-cell embryo biopsy similar to that used in preimplantation genetic diagnosis, that does not interfere with the developmental potential of embryos.”

31 Ogilvie, C.M. et al., J Histochem Cytochem, 2005 Braude, P. et al., Nat Rev Genet, 2002

Potential Major Advance Human embryonic stem cell lines derived from single blastomeres Klimanskaya I, et al. Nature 2006; 444:481-485

We report a series of 10 separate experiments demonstrating that hES cells can be derived from single blastomeres. The ES cells generated form derivatives from all three germ layers in vitro and in teratomas in vivo. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many…..

Alternatives to generating ES cells from human embryos To avoid using human embryos to harvest the inner cell mass for ES cell generation, one could use nuclear transfer technology to generate ES cells.

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Nuclear transfer technology to generate human ES cells Nucleus of Adult cell Nuclear Transfer

Donor Oocyte

Donor Oocyte

Inner cell mass Culture & differentiate cells in vitro

Human ES cells

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Nuclear transfer technology to create histocompatible tissues Cell transplant or Bioengineer a heart

Nucleus of Adult cell Inner cell mass Nuclear Transfer

Donor Oocyte

Culture & differentiate cells in vitro

Donor Oocyte

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Good news/Bad news with SCNT Good news: This would constitute essentially an autologous transplant of immunologically “self” tissue Bad news: This would constitute essentially an autologous transplant of tissue with the same genetic predisposition to the tissue and organ dysfunction that led to primary organ failure

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Good news/Bad news with SCNT Given that SCNT is not safe for the creation of children (extremely inefficient and generally unsuccessful strategy for creating liveborn animal subjects), reproductive cloning should be banned. If there is no intent (in fact no ability) to create a human, and the technology does not depend on human fertilization, then SCNT may not approach some of the ethical concerns of ES or EG derivation.

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Challenging questions for SCNT Who will donate the eggs? Should they be paid? Who will harvest and regulate the collection of the donated eggs? What kind of screening of the eggs is warranted? Does this technology negatively impact the role and status of women in our society?

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The Promise of Stem Cell Research

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Adult (Somatic) stem cells General properties of adult stem cells: Reside in a quiescent state in the tissue Give rise to differentiated cells of that tissue Are rare and difficult to isolate and culture Fail to proliferate well ex vivo Fail to retain self-renewal ex vivo

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Adult (Somatic) stem cells General properties of adult stem cells: Reside in a quiescent state in the tissue Give rise to differentiated cells of that tissue Are rare and difficult to isolate and culture Fail to proliferate well ex vivo Fail to retain self-renewal ex vivo

The only stem cells that are used as a well studied human cell therapeutic are human hematopoietic stem cells (adult stem cell)

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Source of hematopoietic stem cells for allogeneic transplantation Hematopoietic stem cells for transplantation may be derived from the patients themselves (autologous transplant) or from another person (allogeneic transplant) Only 25-30% of patients requiring a transplant find a fully matched sibling donor…. therefore unrelated donor cells are sought 42

Source of hematopoietic stem cells for allogeneic transplantation An alternate to a related donor involves seeking an unrelated HLA-matched adult donor outside the family The National Marrow Donor Program registry has 7 million potential allogeneic donors 50-60% of patients are unable to find a fully matched donor Non-white donors least successful (lack of genetic heterogeneity due to few non-white donors)

Umbilical cord blood has emerged as an alternate source of allogeneic stem cells

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Source of hematopoietic stem cells for allogeneic transplantation Umbilical cord blood has been successfully utilized as an alternative source of hematopoietic stem cells for related and unrelated donor stem cell transplants. Advantages of cord blood as a stem cell source: no risk to the donor no donor attrition immediate availability lower incidence and severity of GVHD 44

Limitations of cord blood stem cells for allogeneic transplantation Limitations of cord blood as a stem cell source: Limited amount of material (around 80 mL) Delayed neutrophil engraftment (25-32d)[18d] Delayed platelet engraftment (54-85d)[32d] Reduction in graft versus leukemia effect? 45

We need to understand the molecular regulation of ES cell self-renewal and pluripotency ; Can we manipulate stem cell behavior?

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We need to understand the molecular regulation of ES cell self-renewal and pluripotency ; Can we manipulate stem cell behavior?

Induced pluripotent stem cells (iPSCs) By expressing a combination of several different transcription factors in mouse fibroblast cells, several research groups have confirmed that the fibroblast cells become ES-like in appearance, proliferation, differentiation potential, and germline competency. 47

Summary Adult stem cells represent the clonal, self-renewing cells residing in many tissues and organs that maintain the homeostatic cellular requirements of that tissue or organ for the lifetime of the host.

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Summary Adult stem cells represent the clonal, self-renewing cells residing in many tissues and organs that maintain the homeostatic cellular requirements of that tissue or organ for the lifetime of the host. Embryonic stem cells are culture-derived cells that are obtained when the inner cell mass cells are isolated from the early human blastocyst.

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Summary Adult stem cells represent the clonal, self-renewing cells residing in many tissues and organs that maintain the homeostatic cellular requirements of that tissue or organ for the lifetime of the host. Embryonic stem cells are culture-derived cells that are obtained when the inner cell mass cells are isolated from the early human blastocyst. ES cell derivation raises questions of the moral status of the human embryo

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Summary Adult stem cells represent the clonal, self-renewing cells residing in many tissues and organs that maintain the homeostatic cellular requirements of that tissue or organ for the lifetime of the host. Embryonic stem cells are culture-derived cells that are obtained when the inner cell mass cells are isolated from the early human blastocyst. ES cell derivation raises questions of the moral status of the human embryo Research to discover the pluripotent or proliferative properties in ES cells and then applying this data to adult stem cells may permit entirely new paradigms for thinking about disease treatment 51

Summary At present, there is no evidence to support the use of an ES-derived cell type to treat a human disorder, since no safety or efficacy trials in human subjects with ES cells has concluded. The only proven cell therapy for a human disorder is hematopoietic stem cell transplantation. Organ transplantation represents another method for providing cell therapy for human disease. 52

Summary Ongoing research may provide entirely new methods for inducing pluripotent stem cells for human regenerative medicine. The future may include use of small molecules to direct the specific activation, proliferation, and differentiation of tissue specific stem cells for tissue repair and regeneration. 53