Applications in Molecular Biotechnology Dr Eve Lutz Department of Bioscience Stem & Germ Cell Technology

Reading: Textbook:

pp 1308-1312, Alberts, B et al Molecular Biology of the Cell (4th ed) Garland Science (2002) ISBN 0-8153-4072-9, weblink: http://www.classwire.com/garlandscience

Other references: 1. Mansouri, A (2001) Knockout and knock-in animals. Encyclopedia of Life Sciences. 2. Mansouri, A (2001) Mouse knockouts. Encyclopedia of Life Sciences. 3. Odorico, JS, Kaufman, DS & Thomson, JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19, 193-204. (PDF file (Stem Cells Review Paper) posted in Lecture Notes B423, Bruce) Related information:

Focus on Stem Cells News Collection, Nature 20 June 2002 Stem Cell Web Focus, Nature 20 June 2002

Websites: 1. Centre for Genome Research, Edinburgh. 2. ES cell isolation & Embryonic germ cells, Roslin Institute 3. Welcome to the world of stem cells. Stem cells Important Features:

Have the ability to choose between cellular fates o Prolonged self-renewal o Differentiation Regulated by intrinsic signals and the external microenvironment

Stem cells are present in many tissues of adult animals

Important for tissue repair and homeostasis May only be capable of producing a limited number of cell types o Spermatogonial stem cells in the testis Unipotent Produce spermatozoan o Haematopoietic stem cells Multipotent Produce erythrocytes and all the types of white blood cells Present in other organs which normally undergo very limited cellular regeneration or turnover o Brain o Pancreas

Pluripotent stem cells

Derived from germ cells or cells that give rise to the germline Theoretically can give rise to EVERY cell type in the animal body Proliferate indefinitely First recognised in teratocarcinomas

Teratocarcinomas

Bizarre gonadal tumours containing a wide array of tissues derived from the three primary germ layers Contain a large assortment of tissue types o Cartilage o Squamous epithelia o Primitive neuroectoderm o Ganglionic structures o Muscle o Bone o Glandular epithelia

Three types of mammalian pluripotent stem cell lines

Embryonal carcinoma (EC) cells o Derived from testicular tumours (teratocarcinomas) Embryonic stem (ES) cells o Derived from blastocysts (pre-implantation embryos) Embryonic germ (EG) cells o Derived from primordial germ cells of the post-implantation embryo

Use markers to distinguish between pluripotent stem cells and their differentiated derivatives

Cell-surface markers recognized by monoclonal antibodies o Stage-specific embryonic antigens o Tumour-recognition antigens Oct 4 o POU-domain transcription factor o Differentiation associated with down-regulation of Oct4 levels o Downregulation of the Oct4 gene results in differentiation and loss of pluripotent cells

Pluripotency tested in three independent assays 1. 2. 3. Able to differentiate in vitro in a Petri dish Differentiate into teratomas or teratocarcinomas when placed in adult histocompatible or immunosuppressed mice In vivo differentiation when introduced into the blastocoel cavity of a preimplantation embryo o ES and EG cells contribute to every cell type, including the germline o EC cells colonise most embryonic lineages, but generally do not colonise the germline

ES cells

In the unmanipulated blastocyst-stage embryo o Cells of the inner cell mass promptly differentiate to generate primitive ectoderm o Ultimately differentiate during gastrulation into the three embryonic germ layers

Removed from normal embryonic environment

Cultured in the appropriate conditions, ICM cells give rise to cells that proliferate and replace themselves indefinitely Maintain the developmental potential to form advanced derivatives of all three EG layers

Outgrowth cultures give rise to different types of colonies of cells

Those with an undifferentiated phenotype are subcultured onto feeder layers o Mitotically inactivated murine embryonic fibroblasts Can be expanded to establish cell lines that are immortal

Factors required for maintaining pluripotency

Few of the factors that regulate self-renewal are known Maintaining on feeder layers proved critical to keeping stem cells in the undifferentiated state

o o

Provide the factor(s) that suppress differentiation or promotes self-renewal One factor with differentiation-inhibiting activity has been isolated Leukaemia inhibitory factor

LIF

Neuropoeitic and haematopoietic cytokine Activates a heterodimeric receptor o IL6 family of cytokine receptors Receptors associated with tyrosine kinase - JAK One subunit is gp130 o Activate the STAT family of transcription factors Signal Transducer and Activator of Transcription STAT3

Mouse ES cells

Require LIF for undifferentiated proliferation in the absence of feeder cells Have a doubling time of ~12 hours More likely to be related to primitive ectoderm

Mouse ES cell lines

Have been successfully derived from somatic cell nuclear transfer o Genetic material of oocyte is removed o Replaced with an adult cell nucleus o Oocyte is stimulated to develop into an embryo Differentiation o Withdrawal of LIF o Separation of stem cells from feeder cells o Growth of stem cell colonies in suspension Form embryoid bodies Upon dissociation can be plated to yield differentiating cells

Mouse pluripotent stem cells

The use of mouse ES cells has contributed enormously to understanding the events of embryonic, fetal and postnatal development as well as adult homeostasis, not only for mice but for mammals in general Provided valuable starting point for establishing human cell lines

Human ES cells

A useful differentiation culture system to study the mechanisms underlying human development Potential medical benefits

o o

Provide an unlimited supply of tissue for human transplantation Cell based therapy to treat a variety of diseases Parkinsons Disease Diabetes Heart failure

Differences between mouse and human ES cells

Cell morphology o Human ES cells form relatively flat, compact colonies o Easily dissociate into single cells o Mouse ES cells form tighter, more spherical colonies that are more refractory to standard dissociation methods Human ES cells grow more slowly o Population doubling time ~36 hours

Isolation of human ES cells

Requires feeder cells & 20% fetal calf serum Does not require LIF Other factors?

Isolation of EG cells

Both human and mouse cells require the same combination of growth factors o LIF o Kit ligand c-Kit receptor o Basic fibroblast growth factor FGF receptor Once established, no longer require bFGF for growth

Many differentiated cell types can be derived from EG cells

Cardiomyocytes Hematopoietic progenitors Yolk sac Skeletal myocytes Smooth muscle cells Adipocytes Chondrocytes Endothelial cells Melanocytes Neurons Glia Pancreatic islet cells

Primitive endoderm

Build a complete gene expression Road Map

During development, how different cell types o Are formed o survive o proliferate o differentiate o migrate Information will help determine how embryogenesis can go wrong o Study the effects of teratogens o Reprogramming genes that malfunction

In vitro techniques for dissecting developmental processes

Directed differentiation Gene trapping Lineage marking Cell ablation Lineage selection

In vitro culture

Compare downstream gene expression profiles between null mutant and wild-type ES cells o Map the complex network of transcription factors regulating tissue-specific gene expression Provides a unique setting enabling control of the extrinsic cytokine or growth factor environment o Analyse how these factors influence cellular differentiation

In vitro differentiation of ES cells transduced with gene trap vectors

Identifying novel developmentally regulated genes important in tissue-specific differentiation programmes

Developing transplantation therapies

Many potential problems to overcome o Large quantities of stem cells needed o Human ES cells slow growing, differentiate readily and cloning efficiency is very low Be able to differentiate in a controlled manner o Homogenous population of cells o Histocompatible

Be safe and effective

Safety issues

Can cells be derived that are histocompatible with every individual? Will transplanted pluripotent stem cells form tumours or otherwise differentiate inappropriately after transplantation? Infectious agents present in embryo-derived pluripotent cells or acquired by stem cells in feeder-dependent culture o Bovine serum albmumin

Differentiation is a haphazard process

Many types of differentiated cells are produced Isolating specific cell types o fluorescence-activated cell sorting (FACs) o Culture in conditions that favour one cell type over another o Use selectable markers such as green fluorescent protein or resistance to neomycin (G418) Must be expressed from a promoter construct that is introduced by electroporation or transfection into the starting population of pluripotent stem cells Cell- or tissue-specific promoter

Ethical considerations

Pluripotent stem cells are derived from human embryos o 'Spare embryos donated by people undergoing in vitro fertilisation treatment o Donated oocytes fertilised specifically for the purpose of deriving ES cell lines o SCNT embryos

64 human ES cell lines in existence

Policy proposed by President George Bush is to use the existing cell lines for clinical research into stem cell-based therapies NIH not allowed to fund research on cell lines developed after 9 August 2001 cut off date Distribution is limited by several issues o Patenting o Commercial secrecy o Restrictive material transfer agreements

UK policy

Research can be carried out on donated embryos o Limited in number Allows fertilisation of donated oocytes for purpose of the project

Using human SCNT embryos

Principal objection is that it might facilitate reproductive cloning in humans Potential benefit is that they could be made from the patients own somatic cell nuclei o Transplant rejection should not occur Would require a supply of unfertilised human eggs o Inefficient process, requires several eggs to make one SCNT embryo o Egg donation is not without risk o Long waiting lists of women needing donated eggs for treatment of their own infertility problems

Recent studies have shown that adult stem cells have the potential for producing a wider range of cell types than previously thought

Neural stem cells o blood-forming and muscle tissue Mesenchymal stem cells o Neurons o Glia o Smooth muscle o adipocytes

Adult stem cells are somatic cells

No longer capable of making germ cells In the adult animal, only germ cells retain the ability to make a new organism o Property known as developmental totipotency Except in SCNT, somatic cells can not be reprogrammed to allow them to become totipotent Therefore may be unable to make certain cell types required for treatment of some human diseases

Other significant differences

Conditions have not been established to enable indefinite propagation of adult stem cells o Without losing differentiation potential o And maintaining normal chromosome number (karyotype) ES and EG cells can be genetically manipulated using the technique of homologous recombination