question

What is a stem cell niche? Cell types found here?

answer

Stem cell niche- area where stem cells survive and close to vascular beds and supportive tissues True stem cells are only present in stem cell niche with supporting cells

question

Stem cells -potentcy? - what is the function of the product sof self renewal -what is a progenitor cell

answer

Stem cells -pluripotent - important for organism to remain alive. When it divides, it self renews (one cell forms a replica of itself) and the other differentiates to a restricted progenitor cell (that still fontinue to form functional tissue cells of various natures)

question

Progenitor cells (from stem stells) -what can they differentiate into? dedifferientate? -how long do they last

answer

Progenitor cells can still dediffereniate into stem cell and transdiffertiate to other types of stem or progenitor cells. Also note that progenitors can continue to divide for many passges and years in vivo (excellent replication potential)

question

Regenerative medicine and stem cell therapy -what is the purpose of transfer of stem cells or progenitor cells? -give an example using a patient with ocular burns

answer

Regenerative medicine and stem cell therapy - transfer of stem cells or progenitors for the purpose of preventing a disease or changing the stem cells/stem cell niche (i.e. BM transplant; cord blood transplant -example is using a patients own limbal stem cells to cure blindness after infection or ocular burn. This involved a biopsy to create an in vitro culture

question

Stem cell threapy- what is this? What constitutes a stem cell (discuss self rewal) Is stem cell therapy gene therapy?

answer

Stem cell therapy is defined as therapy involving stem cell isolation, culture, storage and cell therapy. Note that a stem cell is a cell that can continuously produce one identical cell (self renmal) and one cell that has slightly different fns. Stem cell therapy is a form of gene therapy

question

Single gene disorders -what are the different therapy options? -is protein replacement therapy or stem cell therapy more cost effective? -Why do pharm companies not invest heavily in these single gene disorders

answer

Single gene disorders and stem cell therapy - you can replace the protein OR you can use stem cell transplant (like bone marrow, cord blood or liver cells with stem cells). -Note that STEM CELL THREAPY IS USUALLY MORE COST EFFECTIVE -Pharm proteins/molecules (like drugs) for single gene disorders will be rare because it is not cost effective to produce these for such a small population

question

Multifactorial disorders (alzheimer or dM)- genes and environment -is genetic treatment difficult? -what does modern therapy aim at?

answer

Multifactoral, common and complex genetic disease (like Alzheimer and DM) -many genes (polygenetic) and factors from environment. -genes and factors are not fully understood, so genetic treatment is not really available - modern therapy focus on reducing signs/symptoms of the disease and reduce risk factors or perform surgery (i.e. giving insulin, avoiding foods)

question

Organ transplanation -what type of therapy is this

answer

type of gene therapy

question

What are the 2 reasons to order an organ transplant for a genetic disease

answer

1- to compensate for organ damage and failure (like PCKD, liver cirrhosis with alpha1AT, CF). 2-to replace a normal organ to prevent damage to other tissues (like hypercholesterlema that is familial).

question

What are the 2 major side effects of stem cell threapy

answer

MAJOR SIDE EFFECTS OF STEM CELL THERAPY 1- IMMUNE REJECTION 2- GVHD

question

Do the 2 major side effects apply to autologous transplantation? is autologous transplant used in genetic disease treatment

answer

Stem cell therapy side effects- immune ejection and GVHD -Does not apply to autologous transplantaction. -However, these are not useful to treat a genetic disease because the stem cells will have the same defect.

question

Stem cell self renewal -what are teh 2 methods of this?

answer

Self renewal- e 1-asymmetric (one daughter stem cell and one differenitaed progenitor like tissue stem cells) 2- symmetrical (2 daughter stem cells, early embryo dev)

question

Stem cells -what is differientiation and maturation

answer

Differentiation and maturation- stem cells form different progenitors with different capabilities

question

Stem cells -why do they migrate and home? -how is migration important during tissue repair for stem cells

answer

Migration and homing -stem cells need to reach the ideal niche to form specialized tissues. This is also important during tissue reapir to insure the damaged site is reached.

question

Stem cells -is the natural state active or quiescence? -when do stem cells divide in adults? -effect of chemo on stem cells? growth facotrs?

answer

Quiescence (resting) - stem cells in tissues are quiet (unlike embryoninc). They only divide in injury or hmoeostatic signals. They RESTIST CHEMO AND RESIST GROWTH FACTORS GOAL IS HOMEOTSASIS

question

Stem cells -when do stem cells undergo apoptosis

answer

Apoptosis is seen during renewal and proliferation to control numbers.

question

Transplant stem cells -how long can they survive post transplant -what fns can transplant stem cells serve

answer

Transplanted stem cells - survive many years after tranplsant if compatible with host. -it can also be used to help recreate the host stem cell niche to protect own host stem cells. (niches are influenced by systemic factors that change with age).

question

Stem cell, niche and transplants -how many stem cells are required for normal fn -can stem cell niche restoration prevent a disease -stem cell niche- role in migration -homing- can we adjust with with therapy

answer

-need 10% of stem cells for normal fn -In some cases, stem cell niche restoration can prevent or restore a disease. -Also note that the stem cell niche is important in homing in or circulating or migrationg stem cells. -We can manipulate homing molecules to help stem cells reach the proper stem cell niche .

question

Stem cells in the bone marrow -what happens to these as you age? -why?

answer

Stem cells decrease in age within bone marrow. This may be due to change in stem cell niche and accumulation of DNA damage in the genome or damage to the mito

question

Organ transplant -does this qualify as stem cell therapy -which cell is essentiatila for survival -does this qualify as gene therapy -thus to increase chance of organ surviival, ____ stem cells (increase/decrease)

answer

Organ transplant is considered a form of stem cell therapy. The success of these will depend on survival of stem cells in the organ. -form of gene therapy -increase

question

Depletion of stem cell reserves, change in stem cell niche and diminished stem cell fn -effect on aging? what other thigns can compound this

answer

Depletion of stem cell reserves, change in stem cell niche and diminished stem cell function is postulated to contribute to ageing particularly under environmental toxicity, stress, infection, inflammation and injury.

question

With aging and disese, tissue stem cells will lose ability to regerneate. Also some tissues and organs simply have a low number of stem stem cells. -what tissue has no stem cells -organs with low regeneration ability (name 3 organs)- number of stem cells here? -what 2 tissues have a robust pool of stem cells allowing regeration

answer

-ovary has no tissue stem cells -organs with low stem cells (brain; heart; kidney) have low regeneration ability due to a limit of stem cells -liver and bone marrow- robust amounts of stem cells

question

Stem cells -how can they maintain life for so long? -is this present in somatic cells -what cell has this same characteristic? how do they escape from stem cells homeostasis?

answer

-Stem cells containe telomerase to maintain length of telomere (on chromatids). -This is absent in somatic cells so the telomere shortens with each cell division until it can no longer divide.this allows organs to be a certain size -cancer cells can maintain telomere length and acquire new properities to escape normal stem cell homeostasis

question

Stem cells vs. cancer cells -which has telomeres -which doesn't maintain homeostasis and get get massive genetic abnormalities

answer

So stem cells and cancer cells- both have telomeres. Cancer cells however, don't maintain homeostasis and continually divide and attain massive genetic abnormalities

question

Discuss stem cells and the maternal/fetus unit

answer

Stem cells persist in mother and children and cause microchimerism. This is 'stem cell therapy' from mother nature.

question

What is HLA-Compatible IVF Embryos

answer

HLA compatible IVF embryo - in cases of bone marrow diseases, hard to find HLA match. Some couples will have fertilization to match the HLA of the afflicting child and deliver another child. Use cord blood and bone marrow from that child for the afflicted child.

question

What can be used from birth to help treat things like burns and corneal repair

answer

Stem cells from cord blood; placentas and amnion stem cells- these can transdifferentiate to other cell types

question

Why is cord blood a good source for stem cells? what disease is it critical to use stem cell therapy before 45 days

answer

Cord blood contains stem cells that are between embroyonic and tissue stem cells. These only need matched for 3 of 6 HLA antigens. This means less rejection and less gvdh. used for Krabbe disease

question

Where are pluripotent stem cells dervied from? Recall these are the best type of cells for stem cell therapy, but limited due to ethical concerns What are iPS cells?

answer

Pluripotent- blastocyts iPSC-induced plurpitotent stem cells- these are reprogramed human somatic cells (like fibroblasts or WBC) that can differentiate into mayn tissue types.

question

How do we program cells to iPSC

answer

1-Viral vectors 2-hormones

question

What is the goal of gene therapy?

answer

-introduce genes to appropriate targets cells to restore normal fn and treat or prevent disease

question

Effective gene threapy -What pieces of genetic material are necessary to replace an affected gene

answer

Effective gene threapy 1- Identify the affected gene. Ovtain the function gene with the promoters, regulators and know the mechanisms needed to enhance the tissue gene expression

question

Effective gene threapy -How do you determine and target the appropriate human somatic target cell/tissue / organ (2 ways to target)

answer

Effective gene threapy -Determine the somatic cell/tissue/organ. 1-Use a stem cell or progenitor that can be transduced with genome integration ex-vivo and transfer back to the patient allowing expression 2-viral vector specific to target cell.

question

What are the 3 types of gene therapy? -which is used for LoF -which is used for dominant negative? or gain of fn? -which is used for SCIF? huntingtons? cancer? CF? hemophilia? DMD

answer

1-Gene supplementation or augmentation - SCID, CF, hemophilia, DMD 2-Gene silencing -Huntington or Cancer 3-Gene repair

question

Gene thearpy- Gene supplementation or augmentation -what types of genetic diseases does this target? what is the goal in % of expression

answer

Gene thearpy- Gene supplementation or augmentation -these are LoF single gene mtuations (SCID) -goal is simply 10% expression -example diseases- SCID, CF, hemophilia, DMD

question

Gene therapy-Gene silencing -what types of genetic disease does this target? -example disease?

answer

Gene therapy-Gene silencing -selectively inhibit gene expression. These are the Gain of function or dominant negative mechanisms -this is like RNAi for Huntington disease or cancers

question

Gene therapy-Gene repair -what is goal

answer

Gene therapy-gene repair -use homologous recombination or mismatch repair to reapir the gene

question

What are the 2 methods used to introduce genes to cells -which is more successful? why?

answer

1-viral vector -most successful. nonviral methods transfer efficiency is very low 2-nonviral physical method

question

Gene introduction- viral vectors -what types of viruses are used (2)? which will integrate into the cell and which will remain as a an extrachromosome? -what can silence expression of viral genomes in both methods -do you target rapid or slowly dividing cells? why?

answer

Viral vector -- viral vectors will bind and enter the cell. This is like retrovirus, adenovirus. -Retrovirus will actually integrate gene into the host. this ensure longterm trangene expression, but epigenetics can sitll silence -Adenovirus will remain as an extra chromosome. (episomes). epigenetics can silence -Problem is that rapidly dividing cells can silence this, so more easier to do in slowly dividing cells

question

Gene introduction- nonviral methods -what type of compoenents are used? -how successful is this?

answer

Nonviral - naked DNA (cDNA with regulatory elements), liposomes, receptor mediates, ecetric are used. -This efficiency however is poor and gene expression is frequently lost.

question

What are the 2 ways to introduce the genome (nonviral or viral vectors) to the target tissues/celsl

answer

1-In vivo -injection of gene therapy agent or IV or spray 2-Ex vivo -correct host tissue issue in vbitro and transfer back

question

Viral vector safety contains -what concern is seen with wild viral vectors when using gene therapy vectors

answer

Wild viral vector contamination may reactivate the gene therapy vector and cause infection.

question

Viral vector safety contains -What immune reactions are you concerned about

answer

Modified virsus and transgene can cause ANAPHYLACTIC SHOCK. (severe immune response)

question

Viral vector safety contains -Risk of cancer? how? -how can we reduce risk of cancer? -what gene therapy caused this in the past

answer

Cancer -can be caused by random integrational mutageneisis (activates a proto-oncogene or inhibits a tumor suppressor with insertion) -thus want site specific integration to minimize risk (case about T cell lymphoma with SCID gene thearpy)

question

What are the 2 types of SCID -give genetic defect of each -lymphoid cells and thymus in each? -how to presumptive dx?

answer

1-Somatic SCID- ADA deficiency. this causes stem cells to malfn. in addition, deoxyadenosine accumulates in lymphocytes and kills cell, causing a lack of b and T cells 2-X SCID- IL2R gamma defect -lymphoid cells scarce in each with small thymus -dx with family history (PCR confirms later)

question

SCID -what are the 3 treatment choices -which is treatment of choice

answer

1-Transplant- BM or cord blood -treatment of choice 2-Gene threapy (with a viral vector) 3-Enzyme replacement

question

SCID -what is the treatment of choice for ADA SCID and X linked SCID? -what are complications of this (2)? -which complication is not seen in SCID? why? -which complication is why we use HLA matching? how else can we try to prevent it

answer

Bone marrow - TOC for both ADA SCID and X linked SCID. Otherwise patient dies in 1 year. 1-Complications are transplant rejections if patient immune system attacks BM stem cells. Prefer to match HLA as close as possible. -not that this is not very common in SCID because patient doesnt have immune system 2-GVHD- graft BM sees the host tissue as foriegn and attacks it -prevent with HLA matching and attempt to deplete T cells prior to therapy

question

SCID -although Bone marrow is the TOC, what is another transplantation method? why?

answer

SCID -Cord blood cells- pref. because frozen and readily available to match. Less chance of immune resonse.

question

SCID -gene therapy- when is it used? -which method is used (viral or nonviral)? which virus for X SCID? -why does the transduced cell survive -what is a risk

answer

SCID -use gene therapy in patients without a good match -use a retrovirus to transduce IL2RG to bone marrow in X SCID ex vivo. -theory is that these transduced cells wil have a survivial advantage -risks- insertional mutagenesis of T cells

question

SCID -enzyme replacement therapy for ADA SCID -compare effectiveness to transplant (cord blood or BM)

answer

use polyehtylene glucool ADA (PEG ADA) -less effective than cord blood or BM transplant

question

Sickle cell disease -what is the pathophys of this? genetic defect?

answer

Sickle cell disease -Caused by beta globin gene with a single base sub of glutamic acid to valine. -This causes the cells to stick together and form a sicke shape. -These will clump, cause vascular ischemia and result in multiorgan disease. -Eventually, hemolytic anemia occurs because the spleen is removing too many cells and it enlarges. look for pain crises

question

Sickle cell disease -how is dx achieved -what is treatment (2)

answer

-Dx with Hb electrophoresis and clinical symptoms -Treatment- bone marrow transplant or cord blood transplant

question

Childhood ALL -neoplasm of what cells? -25% have what mutations

answer

Childhood ALL -Childhood ALL is a neoplasm of B cell stem cells. -Clonal disease with chromosomal translocations that accumulate. -25% are due to TEL-AML1 translocation that is confirmed with PCR.

question

Childhood ALL -What is a risk of 6 mercaptorpurine in treatment of ALL -if patient has a negative TMPT status, what can you suspect

answer

Childhood ALL -6 mercaptorpurine- this causes severe events like bone marrow toxicity and failure. -negative TMPT (thioporuine methyl transferase)-predicts neuropeina

question

Childhood ALL -what is current standrard of therapy for children (especially with low or no TMPT activity)

answer

Childhood ALL -stem cell transplantation using cord blood from unrelated infants.

question

Childhood ALL -Is prenatal diagnosis possible

answer

Childhood ALL -Prenatal diagnosis is acquired. It is not possible to dx.

question

Genetic disease treatment is UNSATISFACTORY 1-knowledge issues 2-prediagnostic fetal damage 3-severe phenotype at diangosis -which does Parkinsons apply to? heart defects? Down?

answer

Genetic disease treatment 1-Not enough of the the genes or pathology is known (i.e. Parkinsons). only 50% of genetic diseases have genes known. 2-Prediagnostic fetal damage- problem exists at the time of dx (hard to prevent), thus need prenatal therapy options 3-Severe phenotype at diagnosis (heart defects, Down)- main treatment is surgery. Further

question

What is an advantages of in vitro MSCs from bone marrow form donors in treatment of CV disease, heart diseases and cartilage damage

answer

In vitro MSCs -less immunogenic with no significant immune problems

question

Gene therapy vs stem cell therapy -which is currently a last resort and how will that change in the future

answer

Gene therapy- las tresoirt. as gene therapies becomes safer and more acceptiable, will become first line threapy

question

ALL immune cell gene therapy -how can you cure this by modifying the cell surface using gene thearpy

answer

-use modified HIV virus T cells with chimeriac antigen receptor (CAR) -this t cell with CD19 will bind leukemia cancer cells and kill them.

question

Hemophilia B gene therapy -how can gene therapy be used here

answer

use AAV8 sero type to Factor IX transgene -achives 10% normal F IX levels, which will prevent bleeding

question

HIV gene therapy -what gene conveys resistance to HIV

answer

-transplant blood stem cells with HIV receptor CCR5 mtuations that means HIV resistance

question

Gene therapy in multifactoral diseases like diabetes and alzheimer -what is the goal of gene therapy here

answer

Effect of one gene is too small -try to find a target in some point of a final common pathway in pathogenesis

question

Gene therapy -why are some tissues (lungs/muscles/brain cells) impeding advancements of gene therapy

answer

difficult to get he genes into the target cells

question

Autologous stem cell and gene therapy -why is this a problem in treatment of genetic diseases? -how can we fix the above problem?

answer

-autologous transplants- will have the same genetic problem -thus need to correct genetic issue with gene repair

question

Prenatal or fetal therapy with stem or gene therapy -2 advantages of this?

answer

1-treat prior to issues development 2-immune tolerance (meaning don't have to match as closely)

question

Prenatal or fetal therapy with stem or gene therapy -which is better in SCID- bone marrow transplant as fetus or postnatal bone marrow

answer

-SCID- bone marrow transplant in fetus is better

question

Prenatal or fetal therapy with stem or gene therapy -what needs to be done to ensure therapy can take place

answer

PRENATAL DIAGNOSIS (PUBS, ultrasound, etc)

question

1-Most gene therapy trials aim at what disease 2-First evere gene therapy for a genetic diseases (name disease)

answer

1-cancer 2-SCID

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