hESC Flashcards
(11 cards)
fertalisation and early embryonic development
Fertilization and Early Embryonic Development
A fertilized cell (zygote) begins to divide through cleavage, forming a series of cells:
2-cell, 4-cell, 8-cell, 16-cell stages (known as blastomeres).
The embryo travels from the oviduct (fallopian tube) to the uterine cavity, which takes approximately 5-7 days.
During fertilization, many sperm cells compete, but only one sperm successfully penetrates the zona pellucida (a glycoprotein coat surrounding the egg).
This triggers a calcium surge, leading to the hardening of the zona pellucida, preventing polyspermy (entry of multiple sperm).
Embryo Development in the Oviduct
As the embryo travels through the oviduct, it undergoes cell division (cleavage), but the overall size remains the same.
Each division produces smaller cells (blastomeres).
Upon reaching the uterus, the embryo forms a blastocyst, which consists of:
Trophoblast cells – will contribute to the formation of the placenta.
Embryonic stem cells (inner cell mass) – will give rise to the fetus.
Blastocyst Hatching and Implantation
The trophoblast cells secrete enzymes that break down the zona pellucida, allowing the embryo to “hatch” out.
Around Day 7, the embryo is free from the zona pellucida, and the trophoblast cells secrete proteins that help the embryo embed into the uterine wall.
Implantation occurs around Day 7, during which the embryo starts to integrate into the maternal tissue.
HESC derivation
Blastocyst Stage:
A blastocyst consists of:
Inner Cell Mass (ICM): Source of embryonic stem cells.
Trophoblast: Gives rise to the placenta.
Isolation of the Inner Cell Mass (ICM):
To obtain stem cells, the inner cell mass needs to be isolated from the blastocyst.
This is done using:
A laser to carefully remove the outer trophoblast layer.
Two glass micropipettes (not plastic) to mechanically separate the inner cell mass from the rest of the embryo.
Culturing the Inner Cell Mass:
The isolated ICM is transferred into a cell culture dish containing:
Feeder cells (typically fibroblasts), which are mitotically inactivated (alive but not dividing).
These feeder cells secrete growth factors that support ICM cell proliferation and differentiation.
Cell Proliferation and Differentiation:
The inner cell mass cells gradually detach from the feeder layer and start to divide and proliferate.
Trophoblast cells also detach and stop contributing to further development.
The embryo secretes proteins that may help break down surrounding cells during the process.
Disaggregation of Cells:
A needle is used to manually separate and disaggregate the initial trophoblastic outgrowth to further isolate stem cells.
Day 6 (before TE removal)
Day 6 (after TE removal)
The terms Day 6 (before TE removal) and Day 6 (after TE removal) refer to the process of deriving human embryonic stem cells (hESCs) from a blastocyst at Day 6 of development, focusing on the removal of the trophectoderm (TE) layer.
Explanation:
Day 6 (before TE removal):
This stage refers to the blastocyst before any intervention, where the trophectoderm (TE) cells are still intact.
The trophectoderm is the outer layer of the blastocyst, which later contributes to the formation of the placenta.
The inner cell mass (ICM), which will eventually give rise to embryonic stem cells, is still enclosed within the trophectoderm.
Day 6 (after TE removal):
At this point, the trophectoderm cells have been removed, usually through mechanical or laser-assisted methods.
This exposes the inner cell mass (ICM), allowing scientists to isolate it for further culturing in order to derive stem cells.
The removal of the TE is crucial to prevent contamination by placental precursor cells and ensure a pure population of pluripotent stem cells.
Purpose of TE Removal:
The goal of removing the TE is to isolate the ICM, as the ICM contains the pluripotent stem cells capable of differentiating into any cell type in the body.
The process is typically performed in stem cell research or assisted reproductive technologies (ART).
hESC lines expression
Human embryonic stem cell (hESC) lines are characterized by the expression of specific pluripotency markers, which confirm their ability to self-renew and differentiate into various cell types. These markers include:
Transcription Factors (Regulate Gene Expression):
Oct3/4 (also called POU5F1): Essential for maintaining pluripotency.
Nanog: Helps sustain self-renewal and pluripotency.
Cell Surface Glycoproteins (Used for Identification):
SSEA-4 (Stage-Specific Embryonic Antigen-4): A surface marker unique to pluripotent cells.
TRA-1-60 and TRA-1-81: Specific cell surface glycoproteins associated with undifferentiated hESCs.
Enzyme Activity Marker:
Alkaline Phosphatase (AP) Activity: A biochemical marker present during the early developmental stage of pluripotent stem cells. Positive AP staining indicates undifferentiated cells.
Conclusion:
If all these markers (Oct3/4, Nanog, SSEA-4, TRA-1-60/TRA-1-81, and AP activity) are positive, it confirms the presence of a valid hESC line.
Differentiation Potential of hESCs
hESCs can be differentiated into the three germ layers, both in vitro (in the lab) and in vivo (inside a living organism), demonstrating their pluripotency:
Ectoderm (Outer Layer): Develops into the nervous system, skin, and hair.
Mesoderm (Middle Layer): Forms muscle, bone, blood, and connective tissues.
Endoderm (Inner Layer): Gives rise to the gastrointestinal tract, lungs, and liver.
If differentiation markers for all three germ layers are detected, it confirms the stem cells’ true pluripotent potential.
mesoderm are positive for smooth muscle actin, endoderm- alpha fetoprotein and ectoderm for biii tubulin
the most common way is to let the hesc spontaneously diffeentiate in the disk in vivo with immonocumpromised mice, do not reject foreign cells. inject pluripotent cells and around 2 months later form teratomas.
in vivo testing
When hESCs are injected into immunocompromised mice, they divide indefinitely under the right conditions.
Over time, the cells begin to differentiate spontaneously into various tissue types, forming teratomas (benign tumors containing tissues from all three germ layers).
Teratoma Formation Proof:
If the dissected teratoma contains tissues representative of the three germ layers, it confirms the pluripotency of the hESCs.
Risks:
If the transplanted cells are not properly controlled, they may become malignant, potentially leading to aggressive cancer-like growths.
Tumor removal or treatment strategies are studied to prevent uncontrolled differentiation.
Newly derived hes cell lines should be karyotyped. Different ways to be checked.
Resolution of karyotyping: how deep we can check mutations??
Chromozome size- 100Mb, 100kb? Conventional g-banding karyotpe- may notice 10mb missing. If less do not notice.
Molecular karyotype resolution 100kb.
Deep sequencing resolution 1bp = 1mm
ethical concerns
Ethical Concerns:
Problem:
One of the major ethical concerns regarding human embryonic stem cell (hESC) research is the argument that it involves the destruction of a potential future baby.
Critics argue that extracting stem cells from an embryo results in its destruction, raising moral and ethical questions.
Evaluation:
However, embryos used for research purposes are typically not viable for transplantation, meaning they would not develop into a healthy baby regardless.
Additionally, advancements have provided alternative ways to obtain hESCs without destroying the embryo.
Solution: Derivation Without Embryo Destruction
In 2007-2008, researchers discovered a method to derive stem cell lines without destroying the embryo, addressing the ethical dilemma.
This method involves the extraction of a single cell (blastomere) from the embryo at an early stage without harming its development
hESC derivation from single blastomere
Method: hESC Derivation from Single Blastomere
Embryo Stage:
At the 8-cell stage, a single blastomere (or sometimes two) can be removed without interfering with the embryo’s further development.
The embryo can continue to develop normally and result in a healthy baby.
Proof of Safety – Preimplantation Genetic Diagnosis (PGD):
This concept is supported by PGD, a procedure routinely performed in families with inherited diseases (e.g., Huntington’s disease, cystic fibrosis).
In PGD:
A single cell is extracted from an embryo for genetic analysis using PCR (Polymerase Chain Reaction), which identifies genetic mutations overnight.
Families can then choose to implant a healthy embryo free of genetic disorders.
The remaining embryos are unharmed and can be implanted successfully.
Applying PGD Techniques to hESC Research:
Scientists can use a similar approach to derive hESCs by isolating a single blastomere without harming the embryo, making the method ethically acceptable.
. hESC derivation from isolated blastomeres: Take one cell and use same procedure
drug development, therapy and regenerative medicine
Drug Development, Therapy and Regenerative Medicine
You cannot use just embryonic stem cells - you must differentiate them. They are pluripotent and can
become anything e.g. pancreatic progenitor cells. Different pathways the cells can take and how to direct them in the right way must be known, but this is complicated, and cells may not respon properly or could go astray. Changing pluripotent embryonic stem cells into the differentiated cell of choice requires a lot of work.
For use in a clinical setting, the cells have to be derived in strictly controlled environment following current good manufacturing practice (cGMP) guidelines and undergo extensive validation. The guidelines are a series of general principles that must be observed during the manufacturing process.
Need to identify dna fingerprint, genomic stability, pluripotency, purity -microbial contaminants, purity - adventitious and retroviruses or other viruses, additional testing.
Do not want to transfer disease to patients? No dirty environment?
Clinical trials with hpsc-based therapies
Geron’s phase 1 clinical trial in spinal cord injury
differentiation protocol problems
Manipulating cell culture conditions by adding or removing various growth factors/ changing a substrate on which they grow would cause embryonic stem cells to lose their pluripotency and undergo differentiation; it is still unknown how to systematically and reliably manipulate the culture conditions in such a way to prevent this - Also, the final product still must be purified in order to be used for therapeutic purposes - differentiated cells varies depending on their typeYield of - Current knowledge: encouraging differentiation toward cells of ectodermal origin e.g. retinal pigment epithelium, oligodendrocytes, yields a much higher percentage of desired cell phenotype than attempts made to differentiate eSCs into cells of endodermal origin e.g. hepatocytes - Embryonic stem cells can proliferate indefinitely so there will be enough starting material to produce a sufficient number of any cell type regardless of how low the yield of particular differentiated cell type is
genomic stability problems
Chromosomal instability is typical for most cells maintained for a long time in vitro as extended culture time often results in chromosomal imbalance and structural chromosomal abnormalities, which may or may not result in undesirable consequences for the recipients of stem cell therapies. Therefore, there is a constant need to improve the cell culture conditions in order to make the cells safe for clinical use. - In spite of all the issues, oligodendrocytes and retinal pigment epithelium derived from eSC’s are already undergoing high-profile, well-controlled clinical trials in the USA and the UK. Depending on their outcome, either way, the attitude toward embryonic stem cells is likely to change. Mesodermal cells, cardiomyocytes, derived from embryonic stemcells recently hit the market as the testing model for drug toxicity
fetal and perinatal stem cells
Obtained from the embryos of terminated pregnancies - Multipotent, must be coerced in cell culture in order to differentiate indefinitely - Often grouped as an adult stem cell as their classification is unclear - Somewhat less controversial than eSC’s but regulations differ between countries - USA where rules about patient safety Frequently used in countries outside of the UK/ - and experimental procedure are less stringent - E.g. treat spinal cord injury/CNS disease in Beijing however no measurable claims about efficacy are available and procedures have not been reviewed by any lead researchers or appropriate ethical committees - Used for treating Batten disease: lack lysosomal enzyme leading to complete retinal blindness by the age of 2, 3 years – vegetative, 4- brain dead Perinatal stem cells Can be divided into three groups based on their origin: the amniotic fluid, the placenta or the umbilical cord. 1. Amniotic fluid - week on and is filtered from bot
totipotent, pluripotent, multi, oligo and unipotent
Stem cells: renew themselves via mitotic cell division and differentiate into a diverse range of specialized cell types - Based on their differentiation potential, mammalian stem cells can be divided into The 5 groups: o 1. Totipotent (omnipotent): cells differentiate into all embryonic and extraembryonic tissues to generate complete and viable organisms e.g. fertilized eggs o 2. Pluripotent: develop into all cell lineages, except those related to extraembryonic tissue. S renew and differentiate into all cells of three germ -elfectoderm from which all organs and tissues layers (mesoderm, endoderm, only natural pluripotent stem cells, develop) e.g. embryonic stem cells are the made; thought to be less powerful -induced pluripotent stem cells (iPSC) are manbut possibly more useful in regenerative medicine o 3. Multipotent: self-renew and differentiate only in closely related family of cells e.g. mesenchymal stem cells; mesenchyme is a part of embryonic mesoderm, consisting of loosely packed, unspecialized cells set in a gelatinous ground substance from which connective tissue, bone and cartilage can be formed o 4. Oligopotent: self-renew and differentiate into close related cell type e.g. haemopoietic stem cells differentiate into myeloid and lymphoid lineages o 5. Unipotent: self-renew and differentiate only into one cell type e.g. muscle stem cell - Stem cells can also be classified according to their origins: embryonic and iPS (pluripotent), fetal and perinatal (multipotent) and adult stem cells (usually oligo- or unipotent.)
