13. Stem Cells and Cell Differentiation

exam 4

Is it ok to use hESCs for stem cell therapy (in religious view)?

• some Christian denominations oppose research because it requires the destruction of a human embryo

• which they consider to be a human life, while others may find its use acceptable

• Judaism and Islam generally support stem cell research and therapy based on the principle of preserving life, particularly for treatments like those derived from adult or umbilical cord blood

Multiple uses for Stem Cells

• Increased Understanding of How Diseases Develop

• Cure diseases

• Test New Drugs for Safety

• Generate New Stem Cells to Replace or Aid Diseased or Damaged Organs

• Research How Certain Cells (e.g. cancer stem cells) develop into
  Cancer

• Regenerative Medicine Applications

• Fix Genetic Diseases in the future

• Tissue Engineering – Organs on a Chip

• Clean Meat Industry

Stem Cell

• A cell that can renew (divide) or differentiate

• Above controlled by
  stem cell “niche”

• Number of doublings influenced by source and type

  • hESCs and iPSCs are immortal, adult sourced (i.e. ad-MSCs) 100 to 200+ doublings (approx.) – more than a typical somatic cell that is regulated by the Hayflick Limit

Adult Stem Cells

• Most popular are adipose (fat) derived mesenchymal stem cells (adMSCs) now as of 11/20/2025 in more than 3000 stem cell therapy trials globally registered with World Health Organization

Fetal Stem Cells

• Amniotic, umbilical cord, placenta

Embryonic Stem Cells

• hESCs and hPSCs (latter – not discussed in text) with hESCs in US clinical trials as of 2010

Induced Pluripotent Stem Cells

iPSCs are not in clinical trials in US but patients being treated in Japan and Australia and elsewhere with 116 clinical trials as of 11/20/2025

Differentiation

• Cell becomes more specialized such as a fibroblast or hepatocyte

• But differentiation can be partial or full so critical and accepted molecular metrics need to be in place to compare, for instance, one iPSC generated hepatocyte to another iPSC generated hepatocyte. RNA-seq is one metric

• Some stem cells have “restricted lineage” and are often called “progenitor” cells because they are limited to only one or two types of cells, while others are totipotent

Transdifferentiation (Direct Reprogramming)

• Ability of a differentiated cell to become another type of differentiated cell without going through an embryonic step (e.g. unlike iPSCs)

• First done experimentally in 1987 but several cells have been generated since that time

Dedifferentiation and Redifferentiation

• Ability of a cell to become more embryonic-like and differentiate into another cell type in vivo

• Chemicals like “reversine” can induce de-differentiation

ex: Eastern Red Spotted Newt

Stem Cell Niche

• Also called the stem cell microenvironment

• Critical to controlling cell division vs differentiation

• Complex and includes

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

Potency

Totipotent

• All cell types

• Highest level of “stemness”

Pluripotent

• Many cell types

• Restricted stemness

Multipotent

• Several cell types

• Stemness even more restricted

Unipotent

• One cell type only

Blastocyst

• late pre-implantation stage embryo

• hESCs originate from inner cell mass

Chimera Test Can Determine if a Stem Cell is Totipotent in vivo

• Legal with mice but not with humans

• Thus, we can never prove that any human stem cell derived or isolated in
  the lab is truly totipotent in vivo in humans

• Only true test of totipotency of a candidate stem cell

• Label test stem cell with GFP (green fluorescent protein)

• Implant GFP-labeled test stem cell in blastocyst and then implant chimeric embryo in surrogate mother

• Now track that GFP labeled stem cell in all tissues and organs of newborn

• mESC (mouse derived) are totipotent but can’t say the same for hESC

How can you tell if a candidate stem cells is Totipotent

• chimera test

Biodistribution and Homing

• Ability of stem cells to find “home” – its targeted tissue

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

• Occurs in vivo: Transplanted XX hearts in XY patients have XY cardiomyocytes upon autopsy (10%) – a clear demonstration of endogenous stem cell homing and repair

Shinya Yamanaka

• Induced Pluripotent Stem Cells (iPSCs)

• won the 2012 Nobel Prize

STAP (Stimulus Triggered Acquisition of
Pluripotency)

• a method reported in 2014 where mature cells were supposedly reverted to a pluripotent or even totipotent stem cell state by simply exposing them to mild stress

• The study, claimed these STAP cells could contribute to all tissues and the placenta in a chimeric mouse, was later retracted and deemed fraudulent due to issues with the data and findings

• The accompanying poll shows the public's initial divided belief in the existence of STAP cells prior to the retraction

Fusogenic

Problem with stem cells

• They can spontaneously fuse with each other forming a tetraploid cell (could generate cancer stem cells)

• When injected into patients mechanical stress can cause fusion

Bioethics

• the norms of conduct

• relative terms and country dependent

Therapeutic cloning

• the production of embryonic stem cells for the use in replacing or repairing damaged tissues or organs

• achieved by transferring a diploid nucleus from a body cell into an egg whose nucleus has been removed

• creating embryo develops under laboratory conditions

• responsible for creating embryonic stem cells to treat diseases such as diabetes and Alzheimer’s disease

Reproductive cloning

• the deliberate production of genetically identical individuals; each newly produced individual is a clone of the original

• creating embryo develops under uterine conditions

• important for harvesting stem cells that can be used to study embryonic development

SCID (Severe Combined Immuno
Deficiency) Mice

• Have no B and T cells and thus have a
  compromised immune system

• Are used for determining if an injected candidate stem cell can differentiate in vivo into a multitude of tissue and cell types in vivo

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

Three Ways to Generate Stem Cells in the Laboratory

• Somatic Cell Nuclear Transfer (SCNT)

• Parthenogenesis (hPSCs)

• Induced Pluripotent Stem Cells (iPSCs)

SCNT (Somatic Cell Nuclear Transfer)

• Could be used for autologous or allogeneic stem cell transplants

• No US federal laws ban therapeutic or reproductive cloning research but some states forbid it. But not allowed clinically in the US

• But is it ethical? A “human embryo” is being created

hPSCs (human parthenogenetic stem cells)

• a type of human pluripotent stem cell derived from an unfertilized egg that has been chemically activated, rather than through fertilization

• share many properties with human embryonic stem cells (hESCs), such as the ability to self-renew and differentiate into all three germ layers,

• potential advantages for cell-based therapies like reduced risk of immune rejection

Benefits of hPSCs for therapeutic cloning

• Only 200 to 300 eggs would be required to generated hPSCs that could match anyone in the world

Limitations of hPSCs for therapeutic cloning

• All alleles will be homozygous because of no sperm thus chance of phenotypic expression of a mutation is high compared to heterozygote

• Not FDA approved in US

• Is it ethical to create a human embryo?

iPSCs (Induced pluripoint stem cells)

• Sir Ian Wilmut cloned Dolly the sheep in 1996 and John Gurdon's was the first to work with frogs

• No “human embryo” created as in SCNT and Parthenogenesis

• Can be autologous or allogeneic

• But potential for teratocarcinomas

• More pluripotent than fat (adipose)-derived adult mesenchymal stem
  cells and easier to procure

RT-PCR (Real Time Reverse Transcription Polymerase Chain Reaction)

a sensitive and fast test used for detecting the presence of specific genetic materials within a sample

Is a teratoma generated by iPSC injection?

• generated by injecting induced pluripotent stem cells (iPSCs) into immunodeficient mice

• standard method to confirm the pluripotency of iPSCs

Teratocarcinoma

• A malignant teratoma that
  originates from embryonic cells or stem cells

• symptoms like a painless, firm lump in the scrotum or, if they grow large, pain and swelling in the abdomen, chest pain, or shortness of breath

• Treatment typically involves a combination of surgical removal, chemotherapy, and radiation therapy

Can iPSCs and hESCs do this too? Are iPSCs
in clinical trials like hESCs?

• not yet reached the clinical trial stage in the United States

• (FDA) has cleared multiple Investigational New Drug (IND) applications for iPSC-derived therapies to enter trials

Promise of iPSCs

• Basic research on differentiation

• Can make patient specific cells of
  individuals carrying genetic defects –useful for drug development

• Source of cells in the future for stem cell therapy

  • Not yet FDA approved but Cynata Therapeutics (Australia) just completed first clinical trials in UK using iPSCs.

• Have proven very useful in tissue
  engineering organoids et..

Parthenogenesis

• Can match to a world population – only 300 eggs required (?)

• But all alleles are homozygous, not heterozygous

• Allogeneic, not autologous like SCNT unless female donated egg

• But is it ethical? A “human embryo” is being created

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

ex: A woman injected with human mesenchymal stem cells (MSCs) near her eyes ended up with bone tissue growing inside her eyelids

Regeneration of the Intestinal Epithelium

• a rapid process driven by intestinal stem cells (ISCs) located in the crypts, which replenish the cells of the villi every 5–7 days

• involves ISCs differentiating into progenitor cells, which then become mature cell types

• The regenerated cells move up the villi, where they perform their function before undergoing programmed cell death at the tips

Hematopoiesis

• process of creating all types of blood cells from hematopoietic stem cells (HSCs)

• crucial for maintaining blood cell homeostasis, as it involves the HSC niche

• The production is tightly regulated by growth factors and cytokines, such as stem cell factor (SCF) and interleukins, and is essential for the body's immune response and oxygen transport

Cord Blood

• “Blood Replacement and Stem Cell Therapy"

• contains Hematopoietic Stem Cells and Mesenchymal Stem Cells, which can be used for blood replacement and regenerative therapies

Cord Blood - Private

• incorporated as a “for profit” organization

• Donors pay an initial fee and a maintenance fee

• Cells not available to the public

• Better if there is a genetic disease in the family and multiple members require the cells

Cord Blood - Public

• Incorporated as a “not for profit” organization

• Available to the public through the National Marrow Donor Program through which cord blood is matched

Benefits of C.elegans

• Easy to grow on agar plates and is a non-pathogenic roundworm

• Comprised of a limited number of cells (about 1000)

• Translucent – can optically section through organism

• Stable mutants of C. elegans are available for study

• All cells have been coded with a cell specific letter/number code

• Cell division/differentiation patterns can be predicted and always follow the same pattern

Contributions of C.elegans

• RNAi was first discovered in C. elegans (Nobel Prize in 2006)

• The apoptotic genes were first identified in C. elegans (Robert Horvitz won the 2002 Nobel Prize for Physiology or Medicine for his work on apoptosis in C. elegans)

• Many genes like the apoptotic genes have mammalian homologs

• First microRNA (miRNA) discovered (Nobel Prize in October, 2024, to Victor Ambros and Gary Ruvkun)

When and How is Organismic
Polarity Established?

• during early development through a process of symmetry breaking,

• guided by cues like secreted proteins, and then maintained by internal cellular mechanisms

• external or internal cues break the initial symmetry, followed by the signaling of membrane-associated receptors, the recruitment and reorganization of the cytoskeleton, and the polarized localization of proteins

C. elegans - par proteins establish polarity

• by forming two opposing cortical domains—an anterior domain with PAR-3, PAR-6, and PKC-3, and a posterior domain with PAR-1 and PAR-2

• initiated after fertilization by the sperm centrosome, which causes a contraction of the actomyosin network that pushes the anterior PARs to the anterior cortex while an independent feedback loop maintains the posterior PARs at the posterior

• established polarity ensures the first embryonic cleavage is asymmetric, leading to daughter cells with different fates and division patterns.