Stem Cell/Cell Differentiation (18)

Dr. Dr. Alfred Cioffi

Catholic priest with 2 PhDs that was in RVB’s class because the pope told him to

Alabama and human blastocyst

Blastocyst was decided to be morally equivalent to a child/person

Wolly Mammoth Meatball

• Used 1 gene from mammoth DNA

• Inserted into sheep cells

• Made meat

Stem Cells in Space

• BioFrabrication Facility (BFF) attempts to print organs/tissues in microgravity

• Human induced pluripotent stem cells (hiPSCs) in space are more similar to tissues in body than on earth (in petri dish/solution)

Immunomodulatory Stem Cells (in COVID-19)

Autologous adipose-derived mesenchymal stem cells (HB-adMSCs):

• were FDA approved for COVID-19 (desperately)

• MSCs have regenerative properties

• Used in rheumatoid arthritis at first

Stem cell

A cell that can divide or differentiate, controlled by a niche, depends on source/type

Immortal cells

hESCs and iPSCs that can replicate indefinitely while retaining their pluripotency.

Adult stem cells

adipose (fat) derived mesenchymal stem cells (adMSCs)

Fetal Stem Cells

Amniotic, umbilical cord, placenta

• Kept incase child has genetic disease

Embryonic Stem Cells

hESCs and hPSCs

Induced Pluripotent Stem Cells

• Not in clinical trials in US

Vivo vs Vitro Quality of Stem Cell Differentiation

Differentiation can be partial or full, so metrics are needed to compare

• vivo is the control (desired behavior)

• vitro is experimental (testing behavior)

• Used to determine niches and how they impact stem cell growth

Progenitor Cells

Have a restricted lineage, limited to only a few amounts of differentiation types

Transdifferentiation (Direct Reprogramming)

Differentiated cells become another type of differentiated cell without going through an embryonic step (ex. fibroblast transformed into a neuron)

• Probably doesn’t happen naturally in vivo

Dedifferentiation/Redifferentiation

Cells become more embryonic-like and differentiate into another cell type (in vivo)

• ex, red spotted newt loses limb, cells dedifferentiate, then redifferentiate into a new limb

• Reversine is a chemical that can induce dedifferentiation

Potency

The ability of a stem cell to differentiate into different cell types, classified as:

totipotent - Can differentiate into all cell types (zygote, fertilized egg)

pluripotent - Many

Multipotent - Few

Unipotent - Singular

Chimera Test

Determines if a stem cell is totipotent in vivo

• Label mouse blastocyst with GFP-label to see if they are totipotent

• Insert into another blastocyst

• Insert this into a surrogate mother

• Progeny should show that every cell in newborns is green

Mouse embryonic stem cells are totipotent

Biodistribution/Homing

Endogenous Mesenchymal Stem cells (MSCs) can find targeted tissue due to factors released (ex. during injury) in vivo

• XX hearts transplanted into XY patients have XY cardiomyocytes showing stem cell homming/repair

Shinya Yamanaka/iPSCs

Shinya Yamanaka showed that taking adult cells and adding factors, you can make stem cells in two ways:

• Adult cells can be used to make organoids/drugs after adding factors

• Use CRISPR-Cas9 to correct genetic problems and make into a transplant

• age of donor not important

• Yamanaka factors (4 genes): OCT3/4, SOX2, KLF4, c-MYC

Stimulus Triggered Acquisition of Pluripotency (STAP)

Fraudulent,

• differentiated cells are treated with acid

• cells revert back into stem cell state

• “Proved” with chimera test on mouse

Fusogenic probelm

Ability of cells to spontaneously fuse forming a tetraploid cells (can generate cancer stem cells)

• When stem cells are injected into patients, the mechanical stress can cause fusion

Therapeutic vs. Reproductive Cloning

• Therapeutic Cloning:

  • Creates embryonic stem cells for tissue/organs repair.

  • Involves nucleus transfer into an enucleated egg.

  • Embryo develops in lab; not implanted.

  • Treats diseases (e.g., diabetes, Alzheimer’s).

• Reproductive Cloning:

  • Produces genetically identical organism (clone).

  • Embryo implanted into surrogate mother.

  • Used to study development or clone animals (e.g., Dolly).

No federal laws banning this

Severe Combined Immuno-Deficiency (SCID) Mice

These mice have no B and T cells (compromised immune system)

• used to determine if an injected candidate stem cell will differentiate in vivo

• used to determine if a candidate human cancer cell can generate tumors in vivo

Somatic Cell Nuclear Transfer (SCNT)

1 of 3 ways to generate stem cells in labs

A method for generating pluripotent stem cells by transferring a somatic nucleus into an enucleated egg cell (autologous or allogenic transplant)

• The egg's cytoplasmic factors reprogram the somatic nucleus to a pluripotent state.

• Can generate embryonic stem cells (hESCs) for research or therapy.

• hESCs created this way may serve as autografts for patients (genetically matched).

• Research is shifting toward induced pluripotent stem cells (iPSCs) due to SCNT’s technical difficulty and ethical concerns.

• Demonstrated by Dolly the sheep (1996) and later Resus monkeys (2018, 2024). Ethically questioned because clones can have a lot of issues

• Cloned resus monkeys are tested for drug toxicity due to their closer genetic similarity to humans and lack of genetic diversity

Parthenogenesis (hPSCs)

1 of 3 ways to generate stem cells in labs

A form of asexual reproduction where an egg develops into an embryo without fertilization, potentially used to create haploid embryonic stem cells, made by chemically activating unfertilized eggs. (Allogenic, not autologous)

• First shown by Loeb in 1913 (osmolarity change in sea urchin eggs, used unfertilized star fish eggs with dilute acid).

• These hPSCs (parthenotes form blastocysts and be used to derive stem cells

• Applications: May treat diseases (e.g., Parkinson's, diabetes, heart/liver disease) using genetically matched cells.

• Benefits (ISCO): 200–300 eggs could yield hPSCs matching world population

• Limitations (RVB):

  • All alleles are homozygous (↑ mutation risk), not heterozygous.

  • Not FDA approved in the USA/Raises ethical concerns over creating human embryos without fertilization.

Induced Pluripotent Stem Cells (iPSCs)

1 of 3 ways to generate stem cells in labs

iPSCs are adult somatic cells reprogrammed to a pluripotent/self-renewal state using 4 genes: OCT3/4, SOX2, KLF4, c-MYC (Yamanaka factors) derived from skin biopsies; patient-specific iPSCs avoid immune rejection, however repress genes that induce specific differentiation pathway

• First shown in 2007 by Shinya Yamanaka (Japan) and James Thomson (USA).

• Reprogramming mimics embryonic stem cell (ESC) gene networks (e.g., OCT4, SOX2, NANOG).

• Can differentiate into mesoderm, endoderm, and ectoderm lineages (e.g., heart, neurons, pancreas).

• More pluripotent than adipose (fat)-derived adult mesenchymal stem cells

• Used for:

  • Disease modeling

  • Drug screening

  • Gene repair

  • Cell-based therapies

• Major breakthrough: avoids ethical issues tied to embryo use, unlike SCNT or hESCs.

Ian Wilmut/John Gurdon

Ian Wilmut cloned Dolly in 1996 (popular)

John Gurdon cloned frogs in 1960 (not popular, but won noble peace prize in 2012)

RT-PCR

Used to show what gene markers are used in differentiation of iPSCs

Teratoma/Teratomacarcinoma (Monster Tumor)

A type of tumor that can arise from pluripotent stem cells, containing a mixture of different cell types and tissues

teratoarcinoma - malignant

Maturation Phase Transient Reprogramming (Cellular Rejuvenation)

• Developed at Babraham Institute (2022): turns back the aging clock of human skin cells by 30 years.

• Uses Yamanaka reprogramming factors (OCT4, SOX2, KLF4, c-MYC), but instead of full reprogramming to stem cells (which takes ~50 days), they paused at 13 days—before cells lost their identity.

• This partial reprogramming reversed aging markers: telomere shortening, genetic instability, misfolded proteins, and epigenetic drift.

• Result: fibroblasts retained their function but regained youthful traits.

Short-Term Reprogramming for Liver Regeneration

• Salk Institute (2022) used a 1-day exposure to Yamanaka factors in mice liver cells (vs. 50 days for full iPSCs).

• Result: younger, rejuvenated liver tissue with enhanced regeneration, no teratomas or cancers were observed—common risks in long-term reprogramming.

• Ongoing research seeks to understand how short-term exposure balances repair vs. cancer risk.

Safety Issues of all Stem Cell Therapies (3 types)

Tumorigenicity:
– Stem cells have long telomeres and can divide many more times
than normal cells. (Telomeres = “mitotic clock”)
– Propensity to form tumors such as teratocarcinomas
– One clinical trial started in Japan overseen by the RIKEN Institute
(later) was stopped after only one patient due to this concern

Immunogenicity:
– Propensity to trigger immune response
– The more frequent the stem cell injections the higher the chance of
immune rejection complications that could include anaphylaxis
– Autologous as well as allogeneic can launch an immune response

Inappropriate differentiation:
– Risk of stem cells differentiating into cells that were not intended
and not native to target organ
– Example: A woman injected with human mesenchymal stem cells
(MSCs) near her eyes ended up with bone tissue growing inside
her eyelids!

Cord Blood

Can be stored in a bank for genetic disease treatment (private or public), can be donated

C. elegans (Robert Horvitz)

Perfect model organism for cell differentiation as it can be easily predicted and has a fully mapped genome.

• easy to grow on agar plates

• non-pathogenic

• translucent

• consists of 1000 cells-ish (each cell can be tracked/coded)

• RNAi (nobel prize 2006), apoptotic genes (nobel prize 2002, Robert Horvitz) were first identified in C. elegans, and microRNA (nobel prize 2024)

Par Proteins

C. elegans explained how polarity starts by establishing asymmetrical distribution in anterior/posterior in cells during early development. These proteins are essential for determining the spatial orientation of cells.

Discovery of lin-4 miRNA in C. elegans (heterochronic mutants)

heterochronic mutant - In wild type cells, B5, differentiates and produces PDNB but not at the right time (lin-4 mutant)

• MicroRNA (miRNA), lin-4, blocks translation of a message

• lin-14 mutant used to find this out

• First miRNA discovered

Reproducing/Stem Cell-Fueled Xenobots

Embryonic cells from two separate frogs were combine, cells reorganize in a new/unusual way, successfully reproducing