Stem Cells/Cell Differentiation

Dr. Dr. Father Alfred Cioffi

Catholic priest with two PhDs who was summoned by the pope to investigate hESCs

Uses of stem cells

cure diseases, replace or aid diseased or damaged cells, test new drugs for safety, research, printing human organs in space

research involving stem cells

how certain cells develop into cancer; regenerative medicine (organs on a chip); fix genetic disease (w/ CRISPR); clean meat industry

issue with using stem cell-derived hepatocytes when testing for drug safety

Warburg effect of hepatocytes

clean hamburger procedure

tissue taken from cow (stem cells) --> stem cells grow into muscle fibers --> meat

BioFabrication Facility

development of artificial capillaries in space from stem cells

-difficult to do under gravity on Earth

engineered heart tissues study

how human heart functions in space

-uses unique human iPSCs

Hope Biosciences

adipose-derived mesenchymal stem cell therapy to relieve cytokine storm caused by COVID

stem cells

cells that can renew and/or differentiate

-controlled by stem cell niche

types of stem cells

adult, fetal, embryonic, induced pluripotent stem cells

number of doublings of stem cells

depend on source and type

-hESCs and iPSCs: immortal

adult-sourced: 200-300 divisions

adult stem cells

undifferentiated cells found among differentiated cells in a tissue or organ

-adipose-derived mesenchymal stem cells are most popular

fetal stem cells

amniotic, umbilical cord, placental

embryonic stem cells

hESCs and hPSCs

differentiation

process in which cells become specialized in structure and function

partial or full differentiation

stem cells can differentiate to a point and remain (full) or reverse themselves (partial)

restricted lineage

stem cells that have a restricted number of cells it can progenerate

types of differentiation

transdifferentiation (direct programming); dedifferentiation and redifferentiation

transdifferentiation

the process by which stem cells from one tissue differentiate into cells of another tissue without an embryonic step

dedifferentiation and redifferentiation

ability of a cell to become more embryonic-like and differentiate into another cell type

-reversine; red-spotted newt

red spotted newt

can regrow limbs and lens of eyes

-dedifferentiation/redifferentiation ability

reversine

drug that can induce dedifferentiation

-only tested in-situ

stem cell niche

specialized compartments within organs in which stem cells reside and receive from surrounding cells signals related to division and differentiation

stem cell niche influences

neighboring cells, extracellular matrix, local growth factors (FGF), physical environment (pH, oxygen tension, pressure)

stem cell potency

the ability of a particular stem cell to generate different types of differentiated cells

-totipotent, pluripotent, multipotent, unipotent

levels of stem cell potency

totipotent: all cell types; highest level of stemness

pluripotent: many cell types; restricted stemness

multipotent: several cell types; stemness even more restricted

unipotent: only one cell type

levels of stem cell potency in development of embryo

oocyte (totipotent) → development into blastocyst → embryonic stem cells (inner cell mass within blastocyst) (pluripotent) → developed epiblast (multipotent) → endoderm, mesoderm; ectoderm

human embryonic stem cells

stem cells derived from the inner cell mass within a blastocyst

chimera test

demonstrates that a candidate stem cell is truly totipotent

-legal with mice but not humans

limitation of chimera test with humans

cannot prove that any human stem cell derived or isolated is truly totipotent

chimera test process

stem cells (in form of embroidered body) --> label a cell with constitutive GFP --> implant into female surrogate embryo --> progeny have green-labeled muscles

biodistribution/homing

how stem cells find its targeted tissue

homing of stem cells to damaged tissue

damaged or compromised tissue releases factors that causes endogenous MSCs to home to damaged site

in-vivo biodistribution experiment

transplanted XX hearts in XY patients have XY cardiomyocytes upon autopsy

STAP cells

Stimulus triggered acquisition of pluripotency

-infamous report published in 2014, but now retracted

STAP cells formation

highly differentiated cells (fibroblasts) --> acid shock --> STAP cell

STAP cells test

chimeric mouse test

GFP mouse --> isolated cells --> acid shock to induce STAP phenomenon --> STAP cells injected into normal mouse embryo --> GFP present in all tissues in mouse

STAP cells test interpretation

STAP can turn into any cells and tissue in a mouse (totipotent)

STAP cells scandal

original study could not be reproduced

-had to be retracted from Nature

-Dr. Sasai committed suicide after retraction.

fusogenic (stem cells)

can spontaneously fuse with each other, forming tetraploid cell

issue with stem cells being fusogenic

could generate cancer stem cells

-injection into patients could cause mechanical stress, fusion

mammalian fusogenic factors

CD44, CD47 (macrophage); CXCR4/SDF1 (osteoblast)

bioethics

the ethics of medical and biological research

therapeutic vs. reproductive cloning

therapeutic: production of embryonic stem cells for the use in replacing or repairing damaged tissues or organs or diseases

reproductive: deliberate production of genetically identical conditions

severe combined immunodeficient (SCID) mice

have no B and T cells (compromised immune system)

-injected with cancerous cells --> tumor easily forms

SCID mice and stem cells

used to determine if injected candidate stem cell can differentiate in-vivo into a multitude of tissue types

ways to generate stem cells in laboratory

somatic cell nuclear transfer (SCNT), parthenogenesis (hPSCs), induced pluripotent stem cells (iPSCs)

somatic cell nuclear transfer

a cloning technique that involves substituting genetic material from an adult's cell for the nucleus of an egg

SCNT process

enucleate egg (remove nucleus) --> introduce new nucleus into egg (somatic nucleus) --> insert into surrogate female --> offspring

John Gurdon

first SCNT experiment (1960)

-created a cloned frog from an enucleated egg containing a newly inserted nucleus from the cell of a different frog

Ian Wilmut

cloned the first sheep in 1997 named Dolly

Little Nicky

first cloned pet (cat)

autograft

transplantation of healthy tissue from one site to another site in the same individual

stem cell autograft

human in need of some therapy donates fibroblasts --> SCNT with donor egg --> blastocyst, hESCs --> autologous cells --> apply to human

egg influence in SCNT observations

has cytoplasmic factors that can influence SCNT

benefits and drawbacks of SCNT

benefits: can be used for autologous transplants

drawbacks: have to do thousands of times to get successful implantation

parthenogenesis

unfertilized birth (virgin birth)

Loeb (parthenogenesis)

sea urchins (need perm to reproduce) --> osmotic shock --> new offspring

starfish eggs --> acid shock --> new offspring

International Stem Cell Corporation

company dedicated to developing & promoting human parthenogenesis as a source of hPSCs

mechanism of International Stem Cell Corporation hPSC production

replicates mechanical action of sperm penetration

ionomycin (calcium ionophore) and puromycin (protein synthesis inhibitor) --> blastocyst, but with hPSCs instead of hESCs

advantages and disadvantages of hPSCs

advantages: 200-300 eggs --> all hPSCs to match world population (could be easily accessed for treatments)

disadvantages: hPSCs are all homozygous alleles

-no wild type alleles to suppress any deleterious mutations

induced pluripotent stem cells

a pluripotent stem cell that was generated by manipulation of a differentiated somatic cell

adult cells --> iPS reprograming factors --> iPS cells

Shinya Yamanaka and John Gurdon

showed somatic human cells can be converted to true stem cells with only four genes

Yamanaka approach

used retrovirus to ferry into adults OCT3/4, SOX2, KLF4, and c-MYC genes

-sources were skin cells from 36 yo female and 69 yo male

Yamanaka and Thomson factors

Yamanaka: Oct4, Sox2, Klf4, c-Myc

Thomson: OCT3/4, SOX2, NANOG, LIN28

James Thomson

derived the first human stem cell line; iPSCs with Shinya Yamanaka

reprogramming/Yamanaka factors activation and inactivation

activation: self-renewed pluripotency

inactivation: repress genes that induce specific differential pathways

limit of iPSC potency

cannot be totipotent because there is no way to tell (chimera test limit on humans)

applications of iPSCs

liver disease patients, Cellular Dynamics, Babraham Institute, liver regeneration in mice, reparation of stroke damage on brain

liver disease patient iPSCs treatment

skin or liver biopsy or blood collection --> reprogramming factors added to collected cells --> patient-specific iPSCS -->

-gene correction (CRISPR) --> repaired iPSCs --> hepatic differentiation --> hepatocytes --> transplantation back into patient

-disease modeling (no gene correction) --> hepatocytes --> drug screening/pathology research --> disease- and patient-specific drugs

Cellular Dynamics

sale of beating cardiomyocytes from iPSCs

Babraham Institute

developed a method to reverse 30 years of skin cell aging

- used Yamanaka reprogramming factors

teratoma

monster tumor

iPSCs advantages and drawbacks

advantages: no human embryo created (less ethically polarizing), can be autologous or allogeneic, more pluripotent than adMSCs and easier to acquire

disadvantages: potential for tetracarcinomas

tumorigenicity of stem cells

have long telomeres and can divide many more times than normal cells

-propensity to form tumors and teratomas

immunogenicity of stem ells

propensity to trigger immune response

-potential anaphylaxis after frequent stem cell injections

inappropriate differentiation

risk of stem cells differentiating into cells that were not intended and are not native to target organ

inappropriate differentiation example

woman injecting hMSCs near eye and ended up with bone tissue growing inside eyelids

safety issues with stem cells

tumorigenicity, immunogenicity, inappropriate differentiation

regeneration of intestinal epithelial cells

Lg5+ stem cell located at bottom of intestinal crypt --> new cells rise and differentiate --> reaches villus to perform functions --> cell death when reaches tip of villus

hematopoiesis

blood cell formation

multipotent hematopoietic stem cells

undifferentiated cells capable of giving rise to precursors of any of the different blood cells

Zebrabow

a transgenic zebrafish

-hematopoietic stem cells that can fluoresce up to 80 different colors so that stem cell fate can be tracked

-fluorescent barcoding

fluorescent barcoding

zebrabow; proteins give off specific fluorescence (way to identify)

umbilical cord blood

hematopoietic stem cell source

-blood banking

umbilical cord blood banks

private: for-profit, cells not available to public, better option if there is a genetic disease in family

public: non-profit, available to public through National Marrow Donor Program

ViaCord

banking cord blood cells

Teratocarcinoma

a combination of embryonic carcinomas and undifferentiated somatic (e.g. skin, muscle, bone, glands) tissues

C. elegans

a free-living transparent nematode used for rapid and effective study of gene function

Robert Horvitz

C. elegans

C. elegans benefits

easy to grow (soft agar), nonpathogenic, ~1,000 cells

number of C. elegans cells benefit

reasonable number of cells to follow from zygote to adult form

C. elegans apoptosis

apoptotic genes were discovered through the study of the organism

-information applied to mammalian homologs

PAR proteins in C. elegans

regulators of cytoplasmic partitioning in the early embryo of C. elegans

-established polarity

founder cells

precursor cells that adhere to like cells and undergo mitosis to form tissues.

-C. elegans

first microRNA discovery

C. elegans heterochronic mutant

-lin4-RNA

xenobots

first reproducing stem cell-fused reproduction

-stem cells push cells together in clump to make C-shaped xenobot