Stem Cells

Division of Animal Sciences

College of Agriculture, Food and Natural Resources
Animal Products and Biotechnology

Lecture 10 - Stem Cells

Date: 2025/09/25

Stem Cells

Overview of Differentiation

  • Cells acquire different properties during differentiation due to changes in the types of RNAs produced.

  • Despite these changes, the DNA in each cell remains unchanged.

  • The changes are primarily caused by:

    • DNA cytosine methylation.

    • Alterations in chromatin structure via histone tail modifications.

  • This process is termed "differentiation."

Specific Transcription Markers by Cell Lineage

  • Specific markers are associated with different stages and cell lineages during embryonic development.

  • Markers:

    • Overlap: CDX2 and OCT3/4

    • 8-cell Stage (E2.5): GATA6, Nanog

    • 16-cell Stage (E3.0): Stage-specific markers continue

    • Blastocyst (E3.5):

    • ICM (Inner Cell Mass)

    • TE (Trophoblast)

    • Totipotent blastomere leads to two lineages:

      • Epiblast

      • Primitive endoderm

      • Trophoblast

    • Key Factors in Early Differentiation:

    • Cdx2

    • Oct4

    • GATA-6

    • Nanog

  • Diagram Reference: Fig. 6-4 illustrates the sequential action of transcription factors during initial cell differentiation in the mouse embryo.

Reprogramming

  • Conceptual analogy: Comparing cell differentiation to a marble rolling down a hill—once a path is chosen, it cannot change.

  • In nuclear transfer experiments, the egg acts as a reprogramming agent, akin to kicking a soccer ball back up the hill, reversing differentiation and returning the nucleus to a totipotent state.

Types of Stem Cells

Embryonic Stem Cells

  • Types within a Mouse's Blastocyst:

    • ICM (Inner Cell Mass)

    • TE (Trophoblast)

  • The outer cells (TE) become the placenta, while the inner cells (ICM) can differentiate into various cells forming the body.

Totipotent Cells

  • Definition: Cells that can form a complete organism and all its tissues, including the placenta.

  • Examples: Zygote and early-stage embryonic cells.

Pluripotent Cells

  • Definition: Cells that can differentiate into any cell type but cannot form the placenta.

  • Originates typically from the 8-cell stage onward, specifically the ICM of a blastocyst.

Multipotent Cells

  • Definition: Cells with a fate determined to differentiate into a specific lineage, existing in adult tissues with high turnover rates.

  • Examples:

    • Hematopoietic stem cells

    • Neural stem cells

    • Mesenchymal stem cells

Unipotent Cells

  • Definition: Cells limited to differentiating into one specific cell type, more restricted than multipotent cells.

  • Examples: Epidermal cells, spermatogonial cells, muscle cells.

Historical Milestones in Stem Cell Research

  • 1981: Establishment of mouse ESCs (Kaufman et al.)

  • 1994: Establishment of Embryonic Germ (EG) cells (Stewart et al.)

  • 1998: Establishment of human ESCs derived from an IVF blastocyst (Thomson et al.)

  • 2004: Establishment of human cloned blastocyst-derived ESCs (Hwang et al.)

  • 2007: Generation of human iPSCs (Takahashi et al.)

  • 2013: First derivation of hESCs by therapeutic cloning (Tachibana et al.)

Embryonic Stem Cells (ESCs) Explained

  • Martin Evans successfully extracted and cultured inner cells from a mouse blastocyst in 1981, leading to:

    • Continuous proliferation similar to cancer cells.

    • Unique ability to differentiate into any cell type (ESCs).

  • Potential applications include inserting specific genes into ESCs and creating genetically altered animals—a process that results in a chimera (an organism composed of cells from two or more different zygotes).

Process to Create Chimeras

  1. Obtain stem cells from an embryo.

  2. Gene transfer into embryonic stem cells in vitro.

  3. Inject modified embryonic stem cells into a blastocyst.

  4. Transfer the blastocyst into a host mouse.

  5. Chimeric mouse is born.

  6. If progeny are genetically altered, changes incorporated into germ line.

Nobel Prize in Physiology or Medicine 2007

  • Awarded to:

    • Mario R. Capecchi (1/3 share)

    • Sir Martin J. Evans (1/3 share)

    • Oliver Smithies (1/3 share)

Human Embryonic Stem Cells (hESCs)

  • First successful establishment in 1998 (Thomson).

  • Characteristics of hESCs:

    • Unlimited self-renewal

    • Normal karyotype

    • Specific antigen expression (unique surface markers)

    • Pluripotency proven in vitro (lab) and in vivo (animal models)

  • Diagram Reference: Steps laid out for creating hESCs through nuclear transfer, focusing on implantation and culture.

Properties of Embryonic Stem Cells

  1. Unlimited self-renewal capacity with normal karyotype.

  2. High nucleus-cytoplasmic ratio.

  3. High telomerase activity.

  4. High alkaline phosphatase activity.

  5. Expression of stage-specific embryonic antigens (SSEA-1, 3, 4).

  6. Germ line transcription factor Oct-4 expression.

  7. Pluripotency: ability to differentiate into the three germ cell layers in vitro and in vivo.

  8. Totipotency: ability to form a whole organism.

Embryoid Body (EB) and Teratoma Formation

  • EB Formation:

    • ES cells cultured in a petri dish without feeder layer for two weeks, then embedded in agarose/paraffin for immunostaining.

  • Teratoma Formation:

    • Dissociated cells injected into the testis of SCID mouse; teratomas formed observed after 7-8 weeks.

  • Analysis: H&E staining performed after 10-13 weeks for validation of teratomas.

Ethical Issues Surrounding hESCs

  • Destruction of embryos (with potential to develop into fetuses) for obtaining hESCs.

  • Ethical controversy concerning whether embryos should be considered human beings.

Limitations and Challenges for Therapeutic Use of hESCs

  • Major risk of immune rejection due to MHC incompatibility when hESCs are transplanted.

  • Challenges in obtaining hESCs that match patient MHC since this requires sperm and egg from different individuals.

  • Limited use in cell-based therapies because of immune rejection risk.

Induced Pluripotent Stem Cells (iPSCs)

  • iPSCs can differentiate into various cell types, while fully differentiated cells cannot.

  • Pluripotency determined by transcription factors (Oct4, Sox2, Klf4, and c-Myc).

  • Efforts in identifying the minimal genes required for converting differentiated cells back to pluripotent states led to essential transcription factors termed OSKM.

Experimental Framework by Yamanaka and Takahashi

  • In 2006, they observed that inserting 24 genes into mouse embryonic fibroblasts resulted in stem-like properties.

  • Gene Removal Process: Methodical removal of genes to isolate essential factors culminating in the identification of the four key transcription factors.

Therapeutic Cloning and Practical Applications

  • Advantages of ntESCs:

    • Pluripotency

    • Genetic identity with patients reduces immune rejection risk.

  • Drawbacks: Ethical concerns, low efficiency, and potential for reproductive cloning among others.

Patient-Derived iPSCs Advantages and Disadvantages

Advantages:

  • No immune rejection due to genetic similarity.

  • No destruction of embryos needed, offering ethical advantages.

  • Capable of disease modeling and personalized medicine strategies.

  • Easy access through somatic cells (e.g., skin or blood).

Disadvantages:

  • Tumorigenicity: potential for teratoma formation.

  • Technical complexities and variable efficiency in full reprogramming.

  • Untested long-term safety and efficacy.

  • High costs associated with production and differentiation.

  • Possible genetic memory affecting differentiation capacity.

Summary of Pluripotent Stem Cell Chimera Rates

  • Various sources indicate the chimeric capabilities of different cell types ranging from mouse ESCs to iPSCs.

  • Table of Chimera Rates highlights several key findings from various studies demonstrating different rates of fetal and neonatal chimerism based on the donor cell types and host embryos.

Naive vs Primed State in Stem Cells

  • Distinctions between naive embryonic stem cells and primed stem cells, crucial for understanding chimera formation and germ-line competency.

  • Key Characteristics:

    • Naive state: susceptible to forming chimeras, LIF-dependent.

    • Primed state: not conducive to germ-line transmission, LIF-independent.