Introduction
Human embryonic stem cells (hESCs) are derived from preimplantation embryos. The success of hESC derivation heavily relies on optimal culture conditions. This chapter explores the fundamental aspects of human embryonic development, the techniques of sequential culture and co-culture, and specific protocols to improve the efficiency of hESC derivation.
Day-by-Day Breakdown
Day 0: Oocyte Retrieval
The human oocyte (egg) is retrieved from the follicle.
It is prepared for fertilization in the laboratory.
Day 1: Fertilization
The fertilization process begins.
Key Observations:
Two polar bodies and two pronuclei appear in fertilized eggs.
Only eggs with these features are considered successfully fertilized.
Day 2: First Cleavage
The embryo undergoes its first division, forming 2-4 cells (blastomeres).
Embryo Evaluation Criteria:
Blastomere Count: Number of cells.
Fragmentation Rate: Percentage of fragmented cell material.
Symmetry: Uniformity in blastomere size.
Compaction: Degree of cell clustering.
Multinucleation: Presence of more than one nucleus in a cell.
Example Classification:
4, 10, III, 2, 0, 1 x 2 (e.g., 4 cells, 10% fragmentation, etc.).
Day 3: Further Cleavage
The embryo now consists of 6-8 cells.
The same evaluation criteria as Day 2 are applied to assess its quality.
Day 4: Morula Stage
The embryo develops into a 16-32-cell structure known as a morula.
Key Features:
Individual cells become indistinguishable due to compaction (close contact).
Morulae are classified as:
Morula: Loosely compacted cells.
Compacted Morula: Highly compacted cells.
Day 5: Blastocyst Formation
Key Developments:
Spaces form between compacting cells.
Two distinct cell groups appear:
Trophoblast: Outer layer of cells, which will form the placenta.
Inner Cell Mass (ICM): Central cells, which will form the fetus.
The embryo is now called a blastocyst.
Day 6: Blastocyst Expansion and Hatching
The blastocoelic cavity (fluid-filled space) enlarges, causing the embryo to grow.
The zona pellucida (ZP):
Thins due to expansions and contractions.
The embryo begins to "hatch" out of the ZP, preparing for implantation.
Summary of Key Stages
Day 0: Oocyte retrieval.
Day 1: Fertilization confirmed with polar bodies and pronuclei.
Day 2: Cleavage into 2-4 cells, evaluation starts.
Day 3: Growth to 6-8 cells, evaluation continues.
Day 4: Morula stage (16-32 cells), compaction occurs.
Day 5: Blastocyst forms with distinct cell groups.
Day 6: Blastocyst expands and begins hatching.
This progression ensures proper embryo development, critical for successful implantation in assisted reproductive procedures like IVF.
Blastocyst Classification
Blastocysts are classified based on their morphological development and progression. Here are the classifications:
Early Blastocyst
Description:
Small spaces start to appear between the compacted cells.
Significance:
Indicates the initial stage of cavity formation within the blastocyst.
Cavitated Blastocyst
Description:
The blastocoelic cavity occupies more than 50% of the total volume of the blastocyst.
Significance:
The embryo is progressing towards expansion.
Expanded Blastocyst
Description:
The blastocoelic cavity enlarges significantly.
The structure differentiates into two main components:
Trophectoderm (TE): A single outer layer of cells that will form the placenta.
Inner Cell Mass (ICM): A compact cluster of cells that will form the fetus.
Significance:
Represents advanced development and readiness for implantation.
Hatching Blastocyst
Description:
The blastocyst begins to break through the outer layer, known as the zona pellucida (ZP).
Significance:
Prepares for implantation in the uterine lining.
Hatched Blastocyst
Description:
The embryo has completely exited the zona pellucida and is fully exposed.
Significance:
Marks the final stage of blastocyst development, ready for implantation.
Summary Table of Blastocyst Classification
Sequential Culture in Embryo Development
Sequential culture is a specialized protocol in IVF (In Vitro Fertilization) used to optimize embryo development by meeting specific metabolic needs at different stages. It involves using distinct culture media tailored for each developmental phase.
Key Components of Culture Media
Glucose, Pyruvate, and Lactate:
Early stages (zygote to 8-cell): High pyruvate and lactate, low glucose.
Later stages (blastocyst): High glucose.
Amino Acids:
Enhance development through all stages.
Specific requirements shift during progression (e.g., cleavage to blastocyst).
EDTA (Ethylenediaminetetraacetic Acid):
Benefits confined to the cleavage stage.
Protein Source:
Traditionally patient serum (5–20%).
Modern media use recombinant human serum albumin (HSA) for consistency and safety.
Sequential Culture Media
Stage 1 Media:
Supports development from zygote to the 8-cell stage.
Stage 2 Media:
Aids development from the 8-cell stage to the blastocyst stage.
Protocol of Sequential Culture
Day 0 (Oocyte Retrieval):
Prepare culture dishes with Human Tubal Fluid (HTF) drops (50 µl) overlaid with oil.
Incubate overnight in a 5% CO₂ incubator.
Day 1 (Fertilization Assessment):
Identify fertilized oocytes with two pronuclei and two polar bodies.
Rinse fertilized oocytes with HTF and transfer to culture drops.
Day 2 (Cleavage Stage Check):
Check for cleavage (2–4 cell stage).
Prepare CCM medium drops (50 µl) overlaid with oil.
Transfer embryos to CCM and incubate overnight.
Day 3 (Cleavage Progression):
Assess for 6–8 cell stage.
Embryos remain in CCM medium until day 5 or 6.
Day 4 (Morula Stage):
Embryos enter the compaction and morula stage.
Continue incubation in CCM medium.
Days 5–6 (Blastocyst Stage):
Assess embryos for morphological classification:
Early blastocyst.
Cavitated blastocyst.
Expanded blastocyst.
Hatching/Hatched blastocyst.
Perform derivation when good-quality blastocysts are achieved
Conclusion
Sequential culture ensures optimal conditions for embryo development by providing stage-specific nutrients and an ideal environment. The transition from one medium to another reflects the changing metabolic needs of the embryo, ultimately improving the success rate of IVF procedures.
Coculture System in IVF
Embryo co-culture is a supportive technique in IVF that involves growing embryos with "helper" cells to enhance their development. These cells create a more natural environment, improving embryo quality and increasing the chances of implantation and pregnancy. It is particularly helpful for patients with multiple failed IVF cycles or poor embryo quality.
Types of Embryo Co-culture
Endometrial Co-culture
Embryos are cultured with endometrial cells from the patient’s uterine lining.
Mimics the natural uterine environment, enhancing development.
Co-culture with Cumulus Cells
Embryos are cultured with cumulus cells, which surround the oocyte and aid in its maturation, ovulation, and fertilization.
Cumulus cells remain viable for up to 10 days in culture.
Protocol for Embryo Co-culture on Human Endometrial Epithelial Cells
Day 0: Oocyte Retrieval
Prepare culture dishes with IVF medium (e.g., Vitrolife).
Add 50 µl culture drops to the dish, overlay with oil, and incubate overnight in a 5% CO₂ incubator.
Day 1: Fertilization Assessment
Check fertilized oocytes for two pronuclei and two polar bodies.
Rinse fertilized oocytes and transfer to IVF culture drops.
Incubate embryos in IVF and CCM media overnight.
Day 2: Cleavage Stage and Co-culture Initiation
Assess embryos for cleavage (2–4 cells).
Transfer a single embryo to a well of a 24-multiwell plate with an endometrial epithelial monolayer.
Add 1 ml of IVF
medium (1:1 ratio).
Day 3: Development Assessment
Evaluate embryos for continued growth.
Transfer embryos to CCM drops and briefly incubate (10–15 minutes).
Replace IVF
medium with 1 ml of CCM medium in each well containing an embryo.
Day 4: Further Assessment
Check embryos for morphological development.
Days 5–6: Blastocyst Formation and Classification
Evaluate embryos for blastocyst stage development.
Classify morphologically as early blastocyst, cavitated blastocyst, expanded blastocyst, hatching, or hatched blastocyst.