BW: 1-3 Tissue Engineering: Stem Cells

How are embryonic stem cells harvested?

- Human ES cells are derived from 4-5 day old blastocyst

2. Oxygen tension (mesoderm differentiation)

- one aspect of the environment that may impact embryogenesis - development competence of mouse oocytes cultured in vitro is significantly improved with low-oxygen conditions (5% oxygen tension) compared to atmospheric conditions (20% O2), suggesting cell initially reside in a low-oxygen environment - human studies indicate that there is low oxygen tension within the feto-placental unit until the start of the second trimester with the establishment of maternal circulation to the placenta

Ethical issues concerning the use of stem cells

- there are some problematic issues relating to research on stem cells that mainly concern origins and methods of stem cell production - small numbers of adult stem cells can be found in human body - best sources of stem cells are fetuses and embryos - fetal stem cell lines can be cultured from cells isolated from aborted fetuses - stem cells from embryos can be isolated from 5-7 day-old blastocysts - the collection of stem cells of both fetal and embryonic origins involves destruction of the "donor" - the fetus or embryo - and this is ethically problematic

Pluripotent stem cells

- true stem cells, with potential to make any differentiated cell in the body - comes from inner cell mass of the blastocyst

Types of scaffolding

Biopolymers --> natural --> synthetic

Risks associated with stem cell therapy (3)

Important reasons why the FDA has only approved a handful of stem cell therapies 1. lack of sterility 2. cancer (there is a risk of runaway growth, or a cancerous growth) 3. limited oversight - many of the public has bought into the hype regarding stem cell therapy, which means there are many clinics willing to perform unapproved treatments that medical authorities do not oversee

Capturing aspects of embryonic development

In vitro ESC differentiation is routinely carried out by forming embryoid bodies (EBs), which are 3D aggregates of ESCs in suspension (the aggregated of ESCs are taken from a blastocyst

Embryoid body differentiation

- "EB" is broadly used to define aggregates of differentiating pluripotent cells, which are thought to more accurately recapitulate the complex cellular adhesions and signaling of native tissue - when cultured as EBs, the differentiation of ESCs is similar to many of the events during early embryogenesis, including simultaneous differentiation into 1. endoderm 2. mesoderm, and 3. ectoderm lineages

Induced pluripotent stem cells

- 2006, Takahashi and Yamanaka - reported ability to reprogram differentiated somatic cells by retroviral transduction of several embryonic genes - these iPS cells exhibit similar characteristics of ESCs, including self-renewal and differentiation capacities - in subsequent studies, iPS cells have been derived independently by several groups introducing a variety of combinations of (growth) factors including: Oct3/4, Sox2, Klf4, c-myc as well as Nanog and Lin28 in both mouse and human somatic cells

totipotent cells

- able to differentiate into all cell types, including the three germ layers and placental structures (extraembryonic membranes) *the only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage*

3. Fluid shear stress (mesoderm differentiation)

- blood flow may fundamentally impact the development or maturation potential of HSCs (hematopoietic stem cells: give rise to other blood cells) - establishment of circulation (in the mouse) delivers oxygen and nutrients more widely throughout the embryonic tissues and the resulting fluid shear stress or biomechanical forces are important in the formation of the heart and vessels

Embryonic stem cells

- come from embryos - this stage embryo is called a Blastocyst (4-5 days old embryo) - can self-renew forever - are pluripotent - they can differentiate to almost every cell type in the body

Characteristics of ESCs

- defined by ability to self-renew indefinitely, producing more stem cells, and the capacity to differentiate into other cell types - self-renewal is accomplished via both symmetric divisions and asymmetric divisions

Clinical Outlook of ESC cultue

- development of methods for increasing the scale of ESC culture is important to produce clinically relevant yields - in their pluripotent state, ESCs are typically cultured in monolayer, which restricts the number of cells produced, dependent on the culture surface area - suspension culture is amenable to scale-up for larger volume cultures, such as bioreactors - common formats include spinner flasks, rotating wall vessels (high aspect rotating vessel, slow turning lateral vessel) and large-scale bioreactors - so, it is important to consider the impact of the environmental changes necessary for increasing the scale of cultures for clinical settings

Blastocyst structures include...

Trophoblast Blastocoel Inner cell mass

Macroenvironmental controls: oxygen tension, Shear

- manipulation of oxygen concentration and fluid shear stress within PSC differentiation cultures is possible by employing controlled, stirred suspension bioreactors - stirred suspension cultures (SSC) are well suited to control many aspects of the cellular environment. Stirring prevents formation of spatial concentration gradients within the bulk media - the ability to accurately measure culture conditions, such as oxygen tension of pH, allows control processes to maintain constant conditions or to change conditions as desired over time

ESC differentiation in monolayer

- many protocols rely on soluble delivery of factors in monolayer and exploit pathways known to be important in the context of early development - in the absence of anti-differentiation factors such as LIF, mESCs, spontaneously differentiate

Culture conditions for undifferentiated ESCs

- standard culture conditions for mESC growth were largely adapted from established EC cells culture techniques - when maintained as a monolayer in coculture with stromal cells, mESCs can be maintained in an undifferentiated state park - subsequent analysis of the paracrine factors that contribute to the maintenance of pluripotency revealed the key roles of leukemia inhibitory factory (LIF) and bone morphogenetic proteins (BMPs)

Multipotent stem cells

- stem cells that can become a limited number of types of tissues and cells in the body (various cell types in a family of related cells, such as blood cells) - cells that have the capacity to self-renew by dividing and to develop into develop into multiple specialized cell types present in a specific tissue or organ. Most adult stem cells are multipotent stem cells

Mesoderm development

- the lateral plate mesoderm give rise to the heart, blood vessels, and blood cells of the circulatory system as well as to the mesodermal components of the limbs - some of the mesoderm derivatives include the muscle, the muscles of the tongue, connective tissue, skin, bone, and cartilage, endothelium of blood vessels, RBCs, WBCs, and microglia, teeth, the kidneys, and the adrenal cortex

Adult stem cells

- undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues - the list of adult tissues reported to contain stem cells is growing and includes bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelia of the skin, and digestive system, cornea, retina, liver, and pancreas - to be classified as an adult stem cell, the cell should be capable of self-renewal for the lifetime of the organism

Stem cells

- undifferentiated or partially differentiated cells - can differentiate into various types of cells and proliferate indefinitely to produce more of same stem cell

Key ethical questions of stem cell collection

1. The blastocyst used in stem cell research is microscopically small and has no nervous system. Does it count as a "person" who has a right to life? 2. What do various religions say about when personhood begins? Does science have a view on this? 3. In a society where citizens hold diverse religious views, how can we democratically make humane public policy?

Sources of stem cells

1. bone marrow 2. peripheral blood -- PBSCs are easier to collect than bone marrow stem cells, whcih must be extracted from within bones. This makes PBSCs a less invasive treatment option that bone marrow stem cells 3. umbilical cord blood

Tissue engineering

BME discipline that uses combination of cells, engineering, materials methods, and suitable biochemical and physiochemical factors to restore, maintain, improve, or replace different types of biological tissues

Inner cell mass

a group of approximately 30 cells at one end of the blastocoel - forms 3 germ layers that form all embryonic tissues (endoderm, mesoderm, ectoderm)

Natural scaffolds

chitin, collagen, gelatin, albumin, hyaluronic acid, chitosan

3 embryonic germ layer characteristics

embryonic development is recapitulated within the EB, wherein the cells of germ layers develop and subsequently differentiate into committed cell types, including neurons, glia, skeletal and cardiac muscle cells, hematopoietic cells, hepatic cells, and insulin-secreting (pancreatic) cells

Trophoblast

outer layer of cells that surrounds the blastocyst and forms the placenta

Synthetic scaffolds

polyesters, polylactone, polypropylene fumarates, polyanhydride polyurethane, polyphosphazen

1. Endogenous tissue induction (mesoderm differentiation)

soluble signals direct mesoderm induction and development in the embryo, and consequently PSC differentiation strategies have typically involved the exogenous addition of these cytokines to direct mesoderm differentiation

Process to remove stem cells

the Blastocyst is opened and the stem cells are removed with a pipette

Blastocoel

the hollow cavity inside the blastocyst that will form the body cavity

types of stem cells

totipotent, pluripotent, multipotent, unipotent

Components of tissue engineering

1. cells (differentiated, adult stem cells, or embryonic stem cells) 2. scaffolds (hydrogels, nanofibrous, self-assembling, or solid freeform fabricated scaffolds) 3. bioreactors (dynamic cell seeding, improved mass transfer, mechanical stimuli) 4. signals (small molecules, growth factors/polypeptides, nucleic acids like DNA and siRNA, and antisense oligonucleotides)

2 types of stem cells

1. embryonic stem cells (exist only at earliest stages of development) 2. adult stem cells (tissue specific) --> appear during fetal development and remain in our bodies throughout life

3 germ layers

1. endoderm 2. mesoderm 3. ectoderm

Aspects of embryogenesis that influence the expression of Mesodermal inductive factors

1. endogenous tissue induction 2. oxygen tension 3. fluid shear stress

4 unique properties of all stem cells

1. unspecialized 2. proliferation 3. differentiation (can give rise to specialized tissue under certain physiological and experimental conditions) 4. plasticity (cell from one tissue are able to give rise to cell types of completely different tissue

Symmetric division of stem cells

Cell division where two identical Daughter stem cells are formed (of the same fate)

Asymmetric division of stem cells

With each stem cell division, one daughter cell retains its self-renewing capacity (new stem cell) while the other enters a differentiation pathway - ESCs are capable of indefinite self-renewal in an undifferentiated state under defined culture conditions - the highly proliferative nature of undifferentiated ESCs affords derivation of a population of cells from a single-cell clone