SG 102 Final

naturally-occurring stem cells

adult (somatic) stem cells and embryonic stem cells

PSC-based tech (pluripotent stem cells)

• hESC-based transplants
• Induced Pluripotent Stem Cell-based transplants
• Somatic Cell Nuclear Transfer

(regenerative technologies)

stem cell characteristics

1) Reproduces itself indefinitely
2) Is unspecialized (undifferentiated)
3) Under appropriate conditions, can differentiate into specialized cell types

stem cell uses

research & potential therapy
- immunotherapy, pharmaceuticals, replacement organs, etc.

stem cell potency

pluripotent (embryonic) --\> can differentiate into all cell types

multipotent (somatic) --\> can differentiate into multiple cell types

unipotent (somatic) --\> can differentiate into one cell type

locations of somatic stem cells in the body

all over the body, reservoirs for different tissues

somatic stem cells

adult stem cells; undifferentiated cells found among differentiated cells in a tissue or organ

embryonic stem cells

embryonic cells, which can develop into any type of body cell; can go down several different paths

ex. transplants of hematopoietic stem cells

using multipotent blood stem cells in bone marrow
- can get stem cells from someone else (HLA match) --\> chemotherapy --\> reinfusion
- can save stem cells for later --\> chemotherapy --\> reinfusion

Human embryonic stem cells (hESCs)

in lab, sperm and egg join --\> embryo develops for 5-7 days until it becomes a blastocyst --\> inner cell mass removed --\> grow in dish & change culture conditions to cause different cell type differentiation

Donated IVF embryos are often used for hESC research
- Stem cell lines are maintained in culture

clinical trials

‣ Establish scientific evidence by minimizing bias
‣ Legally required for public safety before putting a new drug or treatment on the market

preclinical --\> phase 1-3 (safety & early efficacy) --\> FDA review & approval --\> phase 4 (ongoing monitoring)

- experimental group receives the tested drug/procedure
- control group receives an inactive or standard drug/procedure

- ex. clinical trial with pancreatic cells derived from an hESC line --\> want to introduce functional pancreatic tissue --\> would be in an injected capsule

challenges of hESCs

immune rejection & use of embryos to generate stem cell lines

iPSCs (induced pluripotent stem cells)

Somatic cells that have been genetically altered such that they become pluripotent
- can take off markers of differentiation (upregulate certain stem cell associated genes)
- Pro: No Immune Rejection if autologous (from same patient)

used in macular degeneration clinical trials

iPSC challenges

can the cells become truly pluripotent?
- maybe not
- efficiency of process
- concerns about viral vectors → possible integration into genome

somatic cell nuclear transfer (NT-ESCs)

remove nucleus from a donated egg cell --\> transfer nucleus from patient cells to egg --\> egg cell reprograms the patient's DNA --\> stimulate cell to begin dividing into blastocyst --\> take out cell mass, grow in a dish --\> transfer back to patient

essentially, pulling out the donor's genome and putting in the patient's --\> their own embryonic stem cells

has also been used in reproductive cloning

challenge/questions of stem cell research & therapies

Immune Rejection, Tumorigenicity?, Long-Term Effects?, Need for egg donors , Use of embryos (SCNT & hESCs), Unauthorized stem cell treatments, Scalability/feasibility/Regulation/Access

pluripotent stem cells in research & malignancy

Other uses (aside from drug development and cell therapy):
- toxin screening
- basic research into development
- disease modeling

possible tumorigenicity
- can accumulate mutations by dividing so much
- one study sequenced the genome before insertion and found mutations

14 day rule

international guideline on duration of development allowed in vitro
- 2016: researchers became able to keep embryos (derived from IVF) in culture up to 14 days post-fertilization

recent stem cell research findings

hESCs and iPSCs can be coaxed into self-organizing into neural tissue/brain-like structures
- Implanted human brain organoids showed activity in rat brain

researchers combined mouse ESCs in a device designed to support development → self-organized into synthetic embryos and developed to 8.5d

embryonic stem cell uses

regenerative medicine

embryonic stem cell lines

can create from an existing embryo or through SCNT; either way embryo is destroyed

some cell lines established already

controversy behind embryonic stem cell usage

- some argue embryos deserve human rights --\> when does life start?
- on the other hand, some claim a direct route between research to therapies

history of US law & stem cell research

1995: Congress bans the use of federal funding for any research involving the creation or destruction of embryos (Dickey-Wicker amendment) --\> state only

2001: President Bush's executive order: research permitted only on stem cells lines existing before 9/8/2001 (22 lines)
- but, lines die out

2009: President Obama's executive order lifts the restrictions on funding

CIRM

California Institute for Regenerative Medicine; created after Prop 71 to fund stem cell research

US laws in stem cell research today

Is it legal to use human embryos in research in the US?
▸ YES, federally
▸ NO, in some states (wide variation)

Can scientists receive federal funding for research that involves destroying or creating a human embryo?
• NO (Dickey-Wicker Amendment)

State funding?
• YES, in some states only
• Strong support for SC research in California

qualifications for using spare IVF embryos in research

1. must have been created with intention of attempting pregnancy
2. must have been created without donors
3. must be stored at clinic where created

more embryos are created than needed
- Preimplantation embryos that are never going to be implanted and are ultimately going to be destroyed \= no longer part of a parental project -\> less potential persons
‣ Consented to research by genitors: respect of informed consent

IVF spares for research ethical concerns

Field of fertility is currently very little regulated in the US
- Number of embryos produced is not currently regulated (US)
- Use of young women for egg donation without a complete view of the risks they underg
- Legal and moral status of cryopreserved embryos remains unclear

human embryos in research: balancing ethics

Not a matter of what we can do, rather of what we should do
- what defines human life?

people give legal & moral agreement to research; donate embryos

scientists produce new knowledge/clinical impact; are accountable

human embryos in research: table of opinions

can have full value, intermediate value, or no moral value which influences how you view the status of embryos, their moral consideration, and relevant legal status
- intermediate is what we have today

persons vs. means

Persons have moral rights and are considered to have a value in themselves
▸ Nonhuman animals do not have legal rights (contested for moral rights)
▸ Contrary to a born child, a human embryo doesn't have rights

Non-persons can be used as means towards another end (e.g.,research)

respectful use of embryos

A human embryo can be used as a means

But a human embryo should still be protected
▸ Donor consent
▸ Important research that cannot be done by other means
▸ Specific oversight ("special approval")
▸ The 14-day rule
▸ In US federal funding context, no creation of embryos for research

iPSCs as alternative for embryonic stem cells

Ethical advantages
Ø Does not involve human embryos
Ø Non-invasive procedure

Limitations
Ø Efficiency still limited; need in vivo development
Ø ESCs remain gold standard against which iPSCs stand

rationale for 14 day rule

Last point for twinning to occur (without conjoined twins) (is that equivalent to individuation?)

Not even the founding cells of the nervous system have been specified prior to this stage

There is substantial embryo loss from the time of fertilization up to this point

Until the process of implantation is complete, the embryo has no potential for further development

issues with 14 day rule

differs between countries

Argument of scientists to move the line: much knowledge to be gained

In addition, the current line is not perfect:
▸ Criteria assume that development time is the same for an embryo in vivo and in vitro
▸ Criteria are based on assumptions about embryo development

\=\> Criteria are not natural cut-off points but conventional lines drawn in the process of development

need for 14 day rule (or just a rule)

▸ Basis of public trust
▸ A framework for scientific research
▸ The line should be easy to implement and verify

stem cell therapy scams

there are very few actual therapies that exist; mostly basic research being done; mostly somatic cell treatments

existing stem cell therapies

somatic:
- blood (ex. blood cancer; immunotherapy); skin (burns); eye (corneal injury)

stem cell therapies: FDA regulation

stem cell products are regulated by the FDA (as drugs); but, only have the power to warn, not enforce

The only stem cell products that are currently approved by the FDA are:

1. Blood-forming stem cells (hematopoietic progenitor cells) derived from cord blood --\> Approved for specific uses only (blood diseases)

2. Stem cells used in the context of FDA-approved clinical trials

dangers of stem cell therapy scams

Best-case scenario: it does nothing

Worst-case scenario: it hurts or kills
• Contamined batch
• Malpractice
• Deters from actual medical treatment

should individuals be allowed to use "scams"?

‣ Argument of patient autonomy (but, people aren't actually making an informed choice)

‣ Medically dangerous
‣ Illegal
‣ Preys on people who are in a vulnerable situation (sick people)
‣ Financially vulnerable people
‣ People lacking the social/cultural resources to verify claims

-\> Moral and legal responsibility

Waddington definition of epigenetics

(old understanding)

''the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being.''

thought of epigenetic landscape of differentiation from a stem cell

Riggs definition of epigenetics

(modern definition)

The study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence

epigenome

a multitude of chemical compounds that can tell the genome what to do

epigenomic compounds attach to DNA and modify its function--these marks do not change the DNA sequence. Rather, they change the way cells use the DNA's instructions.

The marks are sometimes passed on from cell to cell as cells divide. They also can be passed down from one generation to the next.

every cell has the same genome; but, each cell only needs a subset (some genes to be expressed & not others)
- epigenome acts like bookmarks

three mechanisms of epigenetic modulation

1. DNA methylation
2. Histone post-translational modifications
3. RNA-based mechanisms

DNA methylation

adds a methyl group to cytosine (DNA base)

usually leads to gene silencing (if methylated, transcription factors cannot bind to promoter since other proteins already bound)

• Transcriptional silencing
• Protecting the genome from transposition
• Genomic imprinting
• X inactivation
• Tissue specific gene expression

DNA methylation & imprinting

only one homologous chromosome expresses a gene because the other is modified (ex. gene only expressed if they come from a female)

About 100 genes in our genome are expressed from only one of the parental chromosome

this is done through methylation

histone modifications (acetylation)

histones have tails that stick out & interact with the DNA (telling it to be expressed or repressed)

either lock up the DNA in a closed package (heterochromatin \= transcriptional repression) OR space out chromatin to be more accessible to transcription factors (euchromatin \= transcriptional activation)
- which one happens depends on when/where modifications are

ex. even serotonin can bind to histones

non-coding RNA modifications

type of RNA that doesn't make protein; regulates DNA expression (ex. if XX, one of Xs is silenced starting through small RNAs)

how the epigenome is interpreted

chromatin associated proteins can...
- add (writers; ex. proteins can add methyl groups)
- remove (erasers; ex. removing methyl groups)
- read (readers; ex. read & need methyl groups to bind)

...different modifications

readers interpret marks and silence/activate genes and/or compact chromatin

what factors can impact the epigenome?

toxins, stress, nutrition, stochasticity (randomness), behavior, etc.

multigenerational vs. transgenerational inheritance

multigenerational exposure \= multiple generations exposed to environmental factor

transgenerational inheritance \= a generation that inherits epigenetic markers without direct exposure

types of generational inheritance of epigenetic markers in rat (example)

multigenerational
- expose F0 pregnant female rat (exposes mother, fetus, and germline)
- F1 fetus was exposed
- F2 was exposed through F1 germline cells

transgenerational
- F3 inherits phenotype with no direct exposure

Dutch Hunger Winter

end of WWII (winter), embargoes created low food availability
- study on men enlisting in the army
- 18 years later, boys were shown to be overweight (that were fetuses during the famine period)

But that's not the end of the story! 70 years later...
- took siblings (one exposed to famine, other born right outside famine period)
- saw clear DNA methylation signature in kids exposed to famine in utero (silencing genes related to insulin, breaking down fat, and obesity)

stress can elicit transgenerational behavioral changes (MSUS study)

subjected mothers to tons of stress (MSUS - multiple, non-regular stressors & maternal separation)
- followed throughout generations, then F3 and F4 given phenotypic battery
- F3 MSUS-exposed lines spent more time floating in forced swim & F3 and F4 had lower latency to enter open arms; less pronounced in F4

how might factors be passed on?

Some "factors" can be passed through the blood and ultimately through the sperm

euchromatin vs heterochromatin

heterochromatin \= transcriptional repression

euchromatin \= transcriptional activation

assisted reproductive technology (ART)

all fertility treatments in which both eggs and embryos are handled (CDC)

In research stages, early clinical, or approved in other countries:
➔ In-Vitro Gametogenesis
➔ Effortless IVF
➔ Uterine Transplants with IVF
➔ Mitochondrial Replacement Therapy (MRT)
➔ CRISPR-Edited Embryos (with IVF)

history of ART regulation

1992 - CDC mandated to collect yearly information about ART cycles performed at US clinics

2016 - introduced soft regulation and more data collection

CDC conducts surveillance, collects data from ART clinics

There are no US laws regulating the practice of IVF or PGD

in vitro fertilization (IVF)

egg and sperm cells are combined outside the body in a laboratory dish (in vitro) to facilitate fertilization

type of ART

IVF process

1. ovarian stimulation
- Typical ovarian cycle (~28 days) yields 1 egg (ovulation \= egg released); IVF typically wants up to 15+ eggs
- this is not a short process; lots of pills and injections
- goals: 1. suppress endogenous hormone cycling; 2. enhance development of multiple mature follicles & collect just before ovulation

2. Retrieval

3. Fertilization
- can use ICSI if sperm motility is cause of infertility; conventional IVF just puts them together in a petri dish

4. Embryo Culture
- cultured to a blastocyst; at this point, researchers could get embryos
- 4.1 Embryo selection and freezing of excess; may include preimplantation genetic testing

5. Embryo Transfer
- transferring embryos into uterus (estrogen and progesterone sometimes used to increase chances of implantation)

6. Pregnancy Test ~2 Weeks Post-Transfer
- 6.1 Optional - selective reductions (e.g., in case of multiple pregnancies; eliminate extra embryo(s))

Preimplantation Genetic Diagnosis (PGD)

In ARTs like IVF, the determination of genetic abnormalities in the embryo before it is transferred to the uterus

PGD is currently unregulated in the United States, while other countries restrict its uses:
➔ UK: Human Fertilization and Embryology Authority sets limits on uses to particular genetic variations; new requests must be reviewed and authorized
➔ France: only allowed for "incurable heritable disease" already present in the family
➔ Gender selection specifically prohibited in many countries (including China, Australia, India,Canada)

Intra-cytoplasmic sperm injection (ICSI)

Inject single sperm into the cytoplasm of single egg
- often done when sperm motility is cause of infertility

what happens to frozen embryos?

Remaining embryos are often frozen and may end up:
○ donated to others for IVF
○ donated to research (e.g.,stem cell research)
○ abandoned (~1/3)

ART data trends in the US

ART cycles - any cycle initiated with intent to transfer an embryo or bank embryos or eggs
- ART cycles have increased over the years; so has banking (freezing) and other cycles

most popular reasons for using are related to infertility

people using donors also on the rise
- One reason people might use egg donors is the decrease in IVF effectiveness with age

egg donorship

- financial incentives may cloud consent
- known short term risks (ovarian hyperstimulation can be fatal)
- possible issues with donor choice & eugenics

justice & ART

a single round of IVF can cost ~$12,000-14,000
- infertility often not covered by insurance

why is number of embryos transferred contentious?

Single embryo transfers have become much more popular over time (as opposed to multiple)

why has this changed?
- health risks for fetuses and pregnant person with multiples

applications of molecular diagnostics

Infectious Disease

Neoplastic Disease

Genetic Disease

Identity Testing

HLA Typing

Pharmacogenetics

techniques applied to molecular genetic testing

• Southern blot
• Dot blot/Reverse dot blot
• *Polymerase chain reaction* - revolutionary for forensic DNA testing (works with very small amounts of DNA)
• RT-PCR
• DNA sequencing - Sanger and NGS
• TaqMan, real-time PCR
• Invader assay
• In situ hybridization
• RNAseq
• Microarray hybridization

PCR DNA typing & saliva on envelopes (+Unabomber)

scams over mail have been solved by sequencing the saliva used to lick the envelope
- Unabomber (thought new tech would be end of humanity; sent mail bombs); got his DNA from some packages and stamps (altho ultimately identified by brother from writing style)

classes of DNA polymorphisms

what DNA testing uses
- RFLP (southern blots)
- VNTR (variable number tandem repeats - very long southern blots)
- STR (short tandem repeats - amplify repeats with PCR)
- SNP (need DNA sequencing)

short tandem repeats (STRs)

sections of a chromosome in which DNA sequences are repeated
- PCR amplifies these for analysis

DNA fingerprinting

analysis of fragments of DNA as a form of identification
- there are DNA probes for polymorphic areas in the genome; each lane is one person (matching)

first convictions using DNA fingerprinting

town with a murder-rape that mandated every adult male in their town get their blood drawn for DNA testing (book written on it popularized DNA fingerprinting) --\> Colin Pitchfork caught for crime

the first US case caught for burglaries & rape

DNA fingerprinting uses

• exoneration (when they aren't a match) --\> innocence project
• Paternity testing
• Establishing zygosity of twins (if defect is genetic + whether twins are identical)
• Determining bone marrow transplant engraftment
• Identifying mislabeled pathology specimens
• Pedigree analysis of animals and animal products
• Establishing identity of rapists, murderers, etc.

Mechanism of DNA fingerprinting

Southern blot in earlier uses
- swab compared to suspect(s)
- can be smeared from degradation (environmental, insect DNA, etc.)

RMNE

random man not excluded (probability random person matches DNA pattern; usually extremely low)
- can never say you're 100% certain because theoretically there could be someone

probability of matching with DNA fingerprinting

with one allele, given around a 1% frequency in given population, already 99% chance you've got the person
- with two alleles, multiply chance together --\> starts getting incredibly small for a random person to match

OJ Simpson case & DNA fingerprinting

gave bad reputation to DNA fingerprinting as unreliable (when it isn't)
- all swabs had OJ's DNA (and some with victims')
- defense argued that database doesn't include enough POC, allele frequency might be higher in Africans (not true --\> African DNA actually most diverse)

chain of custody

the documented and unbroken transfer of evidence (who is in charge of the evidence)

CODIS (Combined DNA Index System)

what if there's no circumstantial evidence pointing to a suspect?

DNA Identification Act of 1994, administered by FBI
- blends forensic DNA technology with computer technology
- digitized DNA fingerprinting results
- who gets put in this database?

cold hit

finding a CODIS DNA database match with no other evidence

How many markers are used in DNA fingerprinting?

FBI requires 13

DNA database laws & arrestee states

all states require sex offenders to be put into CODIS, some require violent crimes/burglary/all felons, some "arrestee states" require anyone that has been arrested (could be released an hour later)
- "DNA drag net"

ethical issues with CODIS use

- who gets put into the database
- do you need an exact match? get some innocent relatives
- use of partial matches (low-stringency)

Grim Sleeper case

CA allowed partial matches to be used (found son of killer)

concerns with low-stringency DNA searches

• Intrusive, invasion of privacy
• Fourth Amendment Issues (ex. using pizza place for DNA)
• Sources of confirmatory DNA specimens
• "False" partial matches
• Challenges personal sexual/paternity information
• CODIS may have a racial bias

Golden State Killer case

Using GEDmatch (commercial DTC genetic test), tracked a distant relative, allowing them to finally find killer

GEDmatch

Use DNA profiles to identify close relatives
- no government oversight/protected by 4th amendment

concerns with forensic use of public genealogy databases

• Intrusive, invasion of privacy
• Unlike CODIS, there is no regulatory/statutory oversight
• DTC genetic tests are not considered "medical" so not protected by HIPAA
• DTC genetic tests are not considered "clinical" so not subject to CLIA regulations for quality assurance
• DTC databases are not protected by the Fourth Amendment
• False results due to partial matches, which (unlike CODIS) are not prohibited by regulations
• False results due to specimen mix-ups and pseudonyms
• Perpetuates the notion that criminality is "genetic"

newborn fingerprinting

genotyping newborns; could be helpful for baby snatching (currently use wristbands)

military genetic testing

DNA testing has been mandatory in the military (idea for there to never again be an unknown soldier(

Romanov family case

shows use of DNA fingerprinting in ancient DNA cases
- only bones left (so had to use mitochondrial DNA)
- mtDNA only inherited from mother
- looked through recessive inheritance of hemophilia to find descendants of the Czar (found a match using British royalty)
- DNA fingerprinting done & Romanovs identified

however, one of the daughters still missing

mass disaster genetics

DNA fingerprinting also used for identification in mass disasters
- ex. commercial airline crash (remaining body parts in coffins); however, dental records usually preferred
- also used for identification in 9/11

Argentina mass graves case

Abuelas de Plaza de Mayo movement - caused many women to be separated from the children when they were killed; tried to match up DNA to return children

DNA fingerprinting & paternity

can be used in paternity testing
- every marker of a child has to be inherited from either the mother or the father
- now offered through consumer services

DTC testing finds nonpaternity 17% of the time

personalized/precision medicine

the use of genetic information to guide drug and dosing selection for subgroups or individual patients in clinical practice

is medicine already personalized?

a common criticism is that medicine is already personal & based on each patient; the Hippocratic Oath teaches this

two arms of precision medicine

gene-level diagnostics (molecular diagnostics)

gene-level therapeutics (gene therapy/gene editing)

Pharmacogenetics

the study of genetically determined variations in drug response
- DNA techniques to predict a patient's drug responses

(pharmacogenomics is the research side)