Stem Cells & Biotechnology – MCDB 370/570 Lecture

These flashcards cover the major concepts, techniques, historical milestones, applications, and ethical considerations presented in the Stem Cells & Biotechnology lecture (MCDB 370/570).

What is the basic definition of a stem cell?

A single, undifferentiated cell that can self-renew and/or differentiate into multiple specialized cell types.

Name the three core properties shared by all stem cells regardless of source.

1) Long-term self-renewal, 2) unspecialized state, and 3) ability to give rise to specialized cell types.

Which type of stem cell has the maximum developmental potential, giving rise to both embryonic and extra-embryonic tissues?

Totipotent stem cells.

At what embryonic stage are totipotent cells naturally present?

From the fertilized egg through the 8-cell stage.

What does pluripotent mean in the context of stem cells?

Cells that can differentiate into any cell of the three germ layers (ectoderm, mesoderm, endoderm) but not extra-embryonic tissues.

Give two examples of pluripotent stem cells found in research.

Embryonic stem (ES) cells and embryonic germ (EG) cells.

Define multipotent stem cells and name one in the body.

Cells whose developmental fate is restricted to a few related lineages; e.g., hematopoietic stem cells in bone marrow.

What is cellular differentiation?

The maturation process by which primitive cells become specialized, functional cell types (e.g., blood stem cell → red cells, white cells, platelets).

In the Gartner Hype Cycle, where does technology often face waning enthusiasm before reaching productive use?

The Trough of Disillusionment.

What is a blastocyst?

A 4–9-day-old pre-implantation embryo (~100–200 cells) consisting of trophectoderm and an inner cell mass (ICM).

From which part of the blastocyst are most embryonic stem cells derived?

The inner cell mass (ICM).

Who first isolated human embryonic stem cells and in what year?

James Thomson (UW-Madison) and John Gearhart (Johns Hopkins) in 1998.

List three molecular markers commonly expressed by embryonic stem cells.

Oct4, Nanog, and Sox-2.

What enzyme activity keeps embryonic stem cells ‘immortal’?

Active telomerase (hTERT).

What in-vitro structure demonstrates the trilineage differentiation capacity of ES cells?

Embryoid bodies (EBs).

Why is teratoma formation considered a hallmark of ES cells?

Because injected ES cells form tumors containing derivatives of all three germ layers, confirming pluripotency in vivo.

Which three scientists shared the 2009 Nobel Prize for discoveries related to telomeres and telomerase?

Elizabeth Blackburn, Carol Greider, and Jack Szostak.

What Nobel-prize-winning technique uses ES cells and homologous recombination to create gene-specific mutant mice?

Knockout mouse technology (Evans, Capecchi, Smithies).

In knockout mouse production, what antibiotic gene is used for positive selection of targeted ES cells?

Neomycin resistance gene (neo).

What drug is used for negative selection against random insertion events in knockout mouse ES cells?

Ganciclovir, acting on the HSV-tk gene in the targeting vector.

What does the phrase “going germline” mean in transgenic mouse work?

Edited ES cells contribute to the gonads so the mutation is heritable.

Which cytokine keeps mouse ES cells undifferentiated in culture?

Leukemia Inhibitory Factor (LIF).

Name one neurological disease targeted by stem-cell-based remyelination strategies.

Multiple Sclerosis.

What cell type is injected in Geron/Asterias’s AST-OPC1 therapy for spinal cord injury?

Partially differentiated oligodendrocyte progenitor cells derived from hES cells.

State two main bioethical or practical problems associated with human ES cells in therapy.

1) Presence of non-human antigens from feeder layers/FBS causing immune rejection; 2) Ethical concern over destruction of human embryos.

Give one culture solution that reduces foreign antigen exposure in hES cell preparations.

Use chemically defined, feeder-free medium (e.g., Millipore’s PluriSTEM).

What are mesenchymal stem cells (MSCs)?

Adult multipotent stromal cells, often isolated from bone marrow or cord blood, capable of forming bone, cartilage, fat, and stromal tissues.

List one advantage and one limitation of MSCs in regenerative medicine.

Advantage: Less ethical concern and easier sourcing. Limitation: Potential immune rejection similar to other transplants.

What is tissue engineering’s basic strategy?

Combine scaffold materials with stem cells to create or repair functional tissues/organs.

Name a Yale scientist noted for tissue-engineered vascular grafts (Humacyte).

Laura Niklason.

What is somatic cell nuclear transfer (SCNT)?

Replacing an oocyte’s nucleus with the nucleus of a somatic cell to reset developmental state.

Distinguish reproductive cloning from therapeutic cloning.

Reproductive cloning aims to create a live offspring; therapeutic cloning generates ES cells for therapy without creating a baby.

Who cloned Dolly the sheep and in what broader category of cloning does this fall?

Ian Wilmut; reproductive cloning via SCNT.

What major SCNT breakthrough occurred in 2013?

Shoukhrat Mitalipov produced human embryonic stem cells from fetal cells via therapeutic cloning.

Define induced pluripotent stem cells (iPSCs).

Pluripotent cells reprogrammed directly from somatic cells using defined factors, without using embryos or oocytes.

List the classical four Yamanaka factors used to reprogram fibroblasts into iPSCs.

Oct4, Sox2, Klf4, and c-Myc.

In which year and by whom were iPSCs first generated?

2006, by Shinya Yamanaka (mouse); 2007 for human cells.

Why are iPSCs considered “autologous friendly”?

They can be derived from a patient’s own cells, reducing immune rejection risk.

What primary cancer concern arose with early iPSC methods?

Random integrating retroviruses could activate oncogenes or disrupt tumor suppressor genes.

Name two non-integrating strategies developed to create safer iPSCs.

Episomal plasmids lost after replication and synthetic modified mRNAs encoding reprogramming factors.

How can genome editing enhance disease modeling with iPSCs?

By creating paired isogenic lines: correcting a patient mutation or inserting it into control lines for direct phenotype comparison.

Outline the basic concept of iPSC-based gene therapy for sickle-cell anemia.

Create iPSCs from patient fibroblasts → repair β-globin gene via homologous recombination → differentiate into hematopoietic progenitors → transplant back to patient.

What is an embryoid body and why is it useful?

A laboratory aggregate of differentiating ES/iPS cells (~10–30k cells) that spontaneously forms derivatives of all three germ layers, allowing study of early development.

Explain the role of LIF in mouse ES cell culture compared with human ES cell culture.

LIF maintains pluripotency in mouse ES cells; human ES cells rely on FGF2 and Activin/Nodal signaling instead.

Which Nobel laureate demonstrated nuclear reprogramming by SCNT in frogs?

John Gurdon (reprogrammed somatic nuclei into enucleated Xenopus oocytes).

What is a chimera in the context of stem cell biology?

An organism composed of genetically distinct cell populations originating from different embryos or species.

Describe one scaffold-free tissue engineering approach mentioned in the lecture.

Generating extracellular matrix (ECM) in culture from MSCs under BMP4 or ascorbic acid to guide later ESC differentiation.

How many embryonic germ layers must be represented to confirm pluripotency in teratoma or embryoid body assays?

All three: ectoderm, mesoderm, and endoderm.

Which U.S. president limited federal funding to existing hES cell lines in 2001?

President George W. Bush.

What is the main therapeutic goal of oligodendrocyte progenitor cell transplantation in spinal cord injury?

To remyelinate damaged axons and improve motor function.

What key culture additive can substitute for mouse feeder layers when maintaining human ES cells?

Conditioned medium supplemented with defined growth factors (e.g., basic FGF) on Matrigel or synthetic matrices.

Explain positive–negative selection in creating targeted ES cell clones.

Positive selection (neo resistance) selects any integration; negative selection (HSV-tk sensitivity to ganciclovir) kills random integrants, enriching homologous recombinants.

Why is telomerase activity critical for stem cell immortality?

It extends telomeres, preventing chromosomal shortening and senescence during numerous cell divisions.

What does ‘episomal’ mean with respect to plasmid-based iPSC reprogramming?

The plasmid remains extra-chromosomal and is eventually lost, leaving no genomic integration.

State one reason embryoid bodies are not found in normal embryonic development.

They are an in-vitro artifact formed by cultured ES/iPS cells aggregating without normal embryonic architecture.

Which molecule(s) are added to differentiation media to generate dopaminergic neurons from hES cells?

Noggin, bFGF, dcAMP, and specific extracellular matrices such as Type IV collagen/fibronectin.

What is the potential clinical use of SCNT-derived ES cells for a diabetic patient?

To create genetically matched pancreatic β-cells for cell replacement therapy.

What is the difference between an embryonic carcinoma (EC) cell and an embryonic stem (ES) cell?

EC cells derive from malignant teratocarcinomas; ES cells are isolated from normal blastocysts and are genetically stable.

Why are feeder-free, xeno-free culture systems important for clinical ES/iPSC applications?

They reduce the risk of zoonotic pathogen transmission and immune reactions to non-human proteins.

Identify one scaffold-based commercial tissue-engineering product mentioned.

Humacyte’s tissue-engineered vascular grafts.

Which growth factor is frequently used to maintain the self-renewal of human ES cells in vitro?

Basic fibroblast growth factor (bFGF or FGF2).

What does the acronym AST-OPC1 stand for in Asterias’s clinical trial?

Astrocyte-like oligodendrocyte progenitor cells, product #1.

Name two diseases for which gene-corrected iPSCs have shown pre-clinical success in mice.

Severe Combined Immunodeficiency (SCID) and sickle-cell anemia.

What is one major immunological hurdle common to both MSC and ES cell therapies?

Potential host immune rejection of transplanted cells/tissues.

Why is c-Myc considered risky in iPSC generation?

It is a potent oncogene that can promote tumorigenesis if reactivated.

Describe ‘matched’ versus ‘unmatched’ iPSC disease modeling.

'Matched' uses isogenic lines differing only in the disease mutation; 'unmatched' compares patient-derived iPSCs to unrelated healthy controls.