Stem cell biology basics By the end of this lecture you will: 1.Understand where stem cells come from 2.Understand the different types of stem cells 3.Understand what stem cells do 4.Understand what stem cell tech has to offer

A life story…

What is a stem cell? Identical stem cells Stem cell SELF-RENEWAL (copying) Stem cell Specialized cells DIFFERENTIATION (specializing)

1 stem cell Self renewal - maintains the stem cell pool 4 specialized cells Differentiation - replaces dead or damaged cells throughout your life Why self-renew AND differentiate? 1 stem cell

Where are stem cells found? embryonic stem cells blastocyst - a very early embryo tissue stem cells fetus, baby and throughout life

Types of stem cell: 1) Tissue stem cells

Tissue stem cells: Where we find them muscles skin surface of the eye brain breast intestines (gut) bone marrow testicles

Tissue stem cells: What they can do MULTIPOTENT blood stem cell found in bone marrow differentiation only specialized types of blood cell: red blood cells, white blood cells, platelets

Types of stem cell: 2) Embryonic stem cells

How are embryonic stem cells grown in the laboratory? Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are isolated by transferring the inner cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer = sticky surface to which they can attach, also release nutrients into the culture medium.

Embryonic stem (ES) cells: Where we find them embryonic stem cells taken from the inner cell mass culture in the lab to grow more cells fluid with nutrients

Over the course of several days, the ESC proliferate and begin to crowd the culture dish. Cells are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months = subculturing. Each cycle of subculturing referred to as a passage.

Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and which appear genetically normal are referred to as an embryonic stem cell line. Once cell lines are established FREEZE CELLS!

What laboratory tests are used to identify embryonic stem cells? –growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term self- renewal. –Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated. –Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells. –presence of a protein called Oct-4, which is typically made by undifferentiated cells. Oct-4 is a transcription factor, helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. –Karyotyping = examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed.

Embryonic stem (ES) cells: What they can do embryonic stem cells PLURIPOTENT all possible types of specialized cells differentiation

neurons grow under conditions B Embryonic stem (ES) cells: Challenges embryonic stem cells skin grow under conditions A blood grow under conditions C liver grow under conditions D ?

What laboratory tests are used to identify embryonic stem cells? –growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term self- renewal. –Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated. –Using specific techniques to determine the presence of surface markers that are found only on undifferentiated cells. –presence of a protein called Oct-4, which is typically made by undifferentiated cells. Oct-4 is a transcription factor, helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. –Karyotyping = examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed.

Pros and cons of each type  Embryonic  Cell lines last and last and last  Pluripotent  Easy to find  Ethical issues - when does life begin?  Adult  Cell lines do not last  Not pluripotent  Hard to locate  No ethical issues

Types of stem cell: 3)Induced pluripotent (iPS) stem cells

Sir John Gurdon

Induced pluripotent stem cells (iPS cells) cell from the body ‘genetic reprogramming’ = add certain genes to the cell induced pluripotent stem (iPS) cell behaves like an embryonic stem cell Advantage: no need for embryos! all possible types of specialized cells culture iPS cells in the lab differentiation

24 candidate genes based on hypothesis that they play pivotal role in maintaining ES cell pluripotency

Fbx15 is specifically expressed in mouse ES cells, but not required = ES cell marker gene Homologous recombination of neomycin r+ cassette into Fbx15 gene via homologous recombination Fbx15

Retroviral infection of 24 factors into mouse embryonic fibroblasts (MEFs) derived from Fbx15 βgeo/ βgeo embryos

0 Colonies when only single factor was transduced Many colonies when all 24 factors were transduced simultaneously

29 colonies, 6 assayed, 4 of which expressed ES cell morphology: -round shape, large nucleoli and scant cytoplasm -similar growth rates (doubling time)

-Similar on a genetic level - Some combination of the 24 transduced factors induced expression of ES cell marker genes in MEF culture

-Removal of individual factors to test their contribution to neomycin r+ colonies -Chose factors which resulted in 0 colonies after 10 days and fewer after 16 days

iPS-MEF3 cells were not healthy

RT-PCR for ES cell marker genes

Upregulated in ES and iPS Upregulated in ES, iPS4/10 > iPS3 Upregulated ES > iPS Global gene expression analyses by DNA microarrays – iPS-MEF4/10 more like ES then MEF

4 factors were sufficient to induce neomycin resistance and ES cell like morphology: Oct3/4 Sox2 Klf4 c-Myc iPS-MEF3 cells are substantially different then iPS-MEFS4/10 iPS-MEFS4/10 are similar but not identical to ES cells: -Similar genetically -Similar morphologically Preliminary Conclusion:

What About Pluripotency?

Teratoma formation is a key indicator of pluripotency cells from all three germ layers are formed

Histology of teratomas derived from iPS-MEF3 cells = undifferentiated cells

Histology of teratomas derived from iPS-MEF4 cells

Trophoblast Endoderm Mesoderm Ectoderm RT-PCR of marker genes from teratomas

iPS-MEF4/10 embryoid bodies began to differentiate into cells from all three germ layers In vitro embryoid body formation Differentiation of embryoid bodies EndodermMesodermEctoderm

Conclusion: the four selected factors could induce pluripotent cells from EMBRYONIC fibroblasts

Can we induce pluripotency on ADULT cells?

Repeated experiments in another cell type: -tail-tip fibroblasts (TTFs) from Fbx15 βgeo/ βgeo which constitutively express GFP iPS-TTFgfp4

RT-PCR ES cell marker

In vitro differentiation

Similar results: -Neomycin resistant colonies -ES cell like morphology -ES cell markers -Teratoma formation -Histology = three germ layers Teratoma histology

-Microinjection of iPS-TTFgfp clone into mouse blastocyst -Implant into a foster mother

-Embryos positive for contribution of iPS-TTFgfp cells up to E13.5 -Histology showed GFP positive cells in all three germ layers

Conclusion: the four selected factors could reprogram adult- terminally differentiated cells to a pluripotent state

Oct3/4 - Mouse embryos that are Oct-4-deficient or have low expression levels of Oct-4 fail to form the inner cell mass, lose pluripotency and differentiate into trophectoderm. Essential transcription factor in maintaining pluripotency (Boyer et al., 2005; Loh et al., 2006) Sox2 - transcription factor that is essential for maintaining self-renewal and pluripotency in undifferentiated embryonic stem cells (Boyer et al., 2005; Loh et al., 2006). c-Myc – may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind their specific target loci. Klf4 - Represses p53 directly, p53 represses Nanog durring ES cell differentiation -> indirectly activates Nanog So why these four factors?

Discussion -Only ~0.02% of transfected cells became ES-like cells, why so low? -Source of iPS cells: contamination? ~0.067% of mouse skin cells are stem cells -Cells induced by 3 factors were nullipotent -Microarray analysis showed cells were not of stem cell origin -Levels of 4 factors required may have a very specific and narrow range -Although mRNA levels were overexpressed in iPS cells, protein levels were similar to ES cells -iPS cells can differentiate in vitro and in vivo despite presence of vectors -Mechanism exists to regulate at protein level -Limited capacity of iPS cells to integrate into normal tissue in vivo ie. No chimera pups -Posttranslational modification differs between iPS and ES cells ie. Methylation -Are these cells caught in an in-between state?

RT-PCR ES cell marker Western Blot

Discussion -Only ~0.02% of transfected cells became ES-like cells, why so low? -Source of iPS cells: contamination? ~0.067% of mouse skin cells are stem cells -ruled out by repeating experiments with bone marrow stroma with high levels of SC -Cells induced by 3 factors were nullipotent -Microarray analysis showed cells were not of stem cell origin -Levels of 4 factors required may have a very specific and narrow range -Although mRNA levels were overexpressed in iPS cells, protein levels were similar to ES cells -iPS cells can differentiate in vitro and in vivo despite presence of vectors -Mechanism exists to regulate at protein level -Limited capacity of iPS cells to integrate into normal tissue in vivo ie. No chimera pups -Posttranslational modification differs between iPS and ES cells ie. Methylation -Are these cells caught in an in-between state?

The future of IPS 1.IPS will offer a pool of genetically viable stem cells for individualized medicine 2.May cure the worst of degenerative diseases -MS -Diabetes -Parkinsons -Paralysis 3.Cells for testing medicine 4.No ethical issues