Stem Cells

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Stem Cells and Self-Renewal

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* specialized cells that continually self-divide and generate progeny cells for
organ formation and maintenance
* found in different tissues at all stages of life, before and after birth
* different types (location in the body, type of cells they can produce)

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Stem cells

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1) Embryonic stem cells: very early embryos
2) Adult stem cells: within tissues of children and adults.

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Embryonic stem cells

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* cultured cell lines derived from the inner cell mass of the blastocyst
* can be grown indefinitely in their undifferentiated state
* capable of differentiating into all cells of the adult body (pluripotency).

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Blastocyst

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Blastocyst (pre-implantation stage), source of embryonic stem cells

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ESCs can only be derived from?

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ESCs can only be derived from cells found in the embryo (ethical concerns).
If a blastocyst is implanted back into the mother for pregnancy, it could develop
into a fetus and then into a newborn baby

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Which SC type demonstrated to produce all the approximately 200 cell types found within the adult body?

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ESCs remain the only SC type demonstrated to produce all the
approximately 200 cell types found within the adult body.

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Human ESCs and IVF

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Not all eggs will fertilize and develop properly into blastocysts during an IVF
procedure. Not implanted blastocysts are frozen for another pregnancy, and
ultimately discarded.
These discarded blastocysts have been used to derive human embryonic stem
cells in the laboratory, after approval of the donors and oversight by the
appropriate IRB or ethics committees

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The value of ESCs for research and therapy

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* Potential to differentiation into all functional cell types.
* Culture methods: ESCs into brain, heart, muscle cells, blood cells, blood
vessels, skin, islet cells and bone cells (3 germ layers).
* Efficiency during derivation, contamination with other cell types, scaling,
dedifferentiation, potential for teratoma formation (benign tumor).
* Research with ESCs will help to discover the factors
necessary for regeneration and repair of tissues (drug discovery).
* Growth of entire hearts, livers and even kidneys. Three-dimensional
matrices (polymer matrices)

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Adult stem cells

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* derived from different parts of the body (svz of brain, bone
marrow, adipocyte)
* have different properties.
* limited number of cell types related to the tissue that
the SCs originally came from.)

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Hematopoietic stem cells

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* adult SCs found mainly in the bone marrow
* form the blood cells
* required for daily blood turnover and for fighting infections.
* easy to obtain, bone marrow aspiration or stimulation with cytokines
(G-CSF) to move into the peripheral blood stream
* HSCs were the first SCs to be used successfully in therapies (blood
cancers, e.g. leukemia) and other blood disorders.

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Mesenchymal stem cells

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* A well-characterized population of adult stem cells.
* Found in the bone marrow, can form fat cells, cartilage, bone, tendon and
ligaments, muscles cells, skin cells and even nerve cells.
* Large quantities.
* Maintained and propagated in culture for long periods of time.
* Can be genetically manipulated (incorporation of DNA sequences)
* Tumorigenic properties if improperly cultured.
* Clinical trials are underway in several clinical centers.

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Umbilical cord blood stem cells

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* Immediately after birth (neonatal stem cells).
* Rich source of hematopoietic stem cells.
* Less mature than adult SCs.
* non-invasive procurement and vast abundance.
* Collection and banked
* Alternative source for the treatment of leukemia and other blood disorders.
(does not produce strong graft-versus-host disease)

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Adult stem cells

In the niche

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* Renew indefinitely
* Mature into more specialized, organ specific cells.
* Asymmetric division (one of the daughter retains the stem cell characteristics,
the other is destined for a limited number of future divisions and will produce the
more organ-specific cells)

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Adult stem cells

However, in the petri dish…

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* Human hematopoietic SCs cannot renew in culture without much success.
* Cocktail of growth factors increase the number of passages of HSCs
* Unlike adult stem cells, embryonic stem cells renew indefinitely in the petri dish

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Somatic cell nuclear transfer (SCNT) or therapeutic cloning.

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*To avoid rejection possibilities: “custom” embryonic stem cells or
patient-specific embryonic stem cells, matching the patient’s immunological profile.
* The DNA from any one cell in the body of a patient (usually a skin or muscle cell)
could be removed and transferred into an unfertilized egg that previously had
its own DNA removed
* In a culture dish, the unfertilized egg is then coaxed into developing as if it
had been fertilized.
* Generation of ESCs (5-6 days)
* The ESCs can now be used to generate cells and tissues for the patient
* Successful in mice and dogs, but not in humans (yet)

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Ethical concerns

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Limited supply of eggs
* animal eggs
*ESC generation can now be done with Federal funding, but donors can
not be compensated and therefore no source of eggs is available; loophole:
however, individuals that are already decided to donate eggs can be
compensated (Columbia University).

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Teratoma potential of ESC lines derived

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Patient-specific (SCNT) hESCs not possible yet.
SCNT could not be done with human ESCs yet (Hwang- South Korea)
Recent experiments showing advances (NYSCF, Eggan); the egg nucleus needs
to be present during the process

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Alternative to ESCs
Induced pluripotent stem cells (iPS cells)
New generation of stem cells

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* Dedifferentiation is possible through cloning.
Dolly the sheep (Wilmut)
* Minimal set of transcription factors are required dedifferentiate an
adult cell (Oct3/4, Sox2, c-myc and Klf4)
• Mouse (Yamanaka, Jaenisch) and human iPS cells (Yamanaka, Thomson)
• Rigorous assays for pluripotency: morphology, expression profile,
methylation pattern, teratomas when ectopically injected in mice, chimeras
in mice, germ line transmission in mice, in vitro differentiation into
several cell types
* Proof of principle experiment: a sickle cell anemia mouse model was
rescued after transplantation with murine hematopoietic progenitors
obtained from iPS cells (Jaenisch)
*Disease modeling in the Petri dish (Long QT syndrome with cardiac myocytes,
ALS with neurons)

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Are iPSCs as good as ESCs?

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• Tumors associated to c-myc re-expression
•  Incorporation of retroviral sequences
•  Random integration of dedifferentiation factors (high clonal variability)
•  Low efficiency (0.1%)
•  Mutations in somatic (skin) cells
•  Epigenetic memory
•  Differentiation potential, variation from colony to colony (even from
technician to technician)
•  immature character of differentiated cells (for example cardiac myocytes)
•  Alternatives: other approaches for reprogramming, replacement with small
molecules, modified-RNA, proteins, miRNA

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Comparative Analysis

Human adult fibroblasts

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Patient-specific iPS cells in humans
No patient-specific (SCNT) ES cells in humans yet

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New concept: transdifferentiation

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No need to reprogram to pluripotent state
This process circumvents teratoma formation

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Transdifferentiation:

Promising approach but still at early stages

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Pancreatic beta cells (insulin-secreting) from pancreatic exocrine cells (Melton)
Cardiac myocytes (Tbx5, Mef2C, Gata4) from cardiac fibroblasts (Srivastava)
Cardiac myocytes (Yamanaka factors), poor serum conditions (no LIF) and
cardiac secreted factors (Ding)
Hematopoieic progenitors from skin fibroblasts (Bhatia)
Motorneurons from fibroblasts (Wernig, Eggan)