stem cell midterm

self-renew

stem cells continuously replace themselves, have a very long life

symmetric division

divide into identical daughter cells

stem cell universally accepted properties

self renew and differentiate into special cell types

asymmetric division

divide into two daughter cells, but one is either created differentiated or becomes differentiated

stochastic differentiation

large cohort become differentiated cells

totipotent

can differentiate into all germ layers plus the placental membranes

pluripotent

can differentiate into all germ layers (endoderm, mesoderm, ectoderm)

multipotent

can differentiate into specific cells within a distinct tissue lineage

unipotent

can differentiate into one specific cell or tissue type

nullipotent

cells that have lot the ability to divide, self-renew, or differentiate, but can be reprogrammed in vitro into pluripotent cells

totipotent cell types

zygote, blastomeres

pluripotent cell types

cells of inner cell mass, embryonic stem cells, induced pluripotent stem cells

multipotent cell types

hematopoeitic, mesenchymal, neural

unipotent cell types

muscle satellite cell, spermatogonia stem cells, epidermal stem cells

nullipotent cell types

red blood cells, mature neurons

what is the ultimate proof of stem cells?

reconstitution, taking stem cells out of one area and putting them into another and being able to see the effect (ie rat chimeras)

pluripotent stem cell evidence

chimera formation and germline transfer

hematopoeitic stem cell proof

reconstitution of lympho-hematopoietic system

Multipotent and nullipotent stem cell evidence

very limited

spermatogonial stem cell evidence

successful engraftment, initiation of spermatogenesis, and restoration of fertility

stem cells need _ to keep their stemness

specialized environment

naturally occuring stem cells

embryonic stem cells from blastocysts, hematopoeitic stem cells, rat nullipotent cells, canine multipotent stromal cells

artificial stem cells

use factors that freeze developmental process or clone via nuclear transfer, embryonic stem cells, induced pluripotent stem cells

how are stem cells named?

by where and when they’re collected

how are pluripotent cells used for transfers?

they are differentiated into needed cell type and then transplanted

evidence based medicine

systematic approach to medicine that uses scientific evidence to help healthcare professionals make decisions about patient care

steps to implement ebm

1. frame the question 2. retrieve best medical evidence 3. critically appraise study for validity 4. relevance to your practice and client case 5. how do we evaluate performance 6. does the treatment align with latest medical guidelines

best types of medical evidence

meta-analysis and systematic reviews

phase 1 trial

safety, to evaluate safety and establish toxicity limit

phase 2 trial

efficacy

phase 3 trial

pivotal, going up against standard of care

combined phase 1 and 2 is mainly about _

safety

open label and single arm trials

everyone gets treatment

blastocyst is _ implantation, epiblast is _ implantation

prior, at time

epiblast stem cells

not as capable of chimera transformation or germline transmission bc of methylation on them from epigenetics

why aren’t lentiviral methods used anymore

after putting gene of interest in cell, could disrupt the gene so use non-integrating vectors instead

highest grade test of pluripotency

germline transmission of PSCs genetics and tetraploid complementation

tetraploid complementation

tetraploid embryo is grown into a complete live animal

2nd highest grade test for pluripotency

chimera

3rd highest grade test for pluripotency

teratoma with all three germ layers

4th highest test of pluripotency

differentation to 3 germ layers in vitro

5th highest test of pluripotency

embryoid body, spontaneous differentation

6th test of pluripotency

markers of undifferentiated PSCs, but can get false positives

hanging drop

where PSCs are forced to aggregate, remove differentiation inhibitors

best species with PSCs evidence

rats and mice

why do canine PSCs have such low efficiency for chimera or germline transfer?

they are epi-blast like

use of ESCs

can knock in/out genes, replace damaged tissue

risks of ESCs

allogeneic use needs immunosuppression, and teratoma risk

uses of iPSCs

can see how diseases form, knockout genes, replace tissue, treat cancer

allogeneic

receive from other than self, risk of immune system destroying

autologous

from self, reduces risk of rejection but increased possibility of diseased cells

universal

gene engineered PSCs that have knocked out incompatible parts to make stealth cells immune system doesn’t recognize

main barriers to stem cells in animals are

policies

policy barriers

reduced animal testing by FDA, and NIH does not count animal clinical trials in clinical trials

first published PSC trial

on making RPEs for people with macular degeneration

dose escalation

give dose, wait and see any adverse reactions, and then increase dose

hematopoetic stem cell

multipotent, self-renewal, functional reconstitution

functional reconstitution

can repopulate hematopoeisis after transplantation

till & mcculloch

first to use stem cells as a regenerative therapy, used HSCs

how did HSCs establish stem-cell concept

mice were irradiated to destroy their HSCs, then donor HSCs were injected, chromosomal markers were used to trace the donor cells, found the same cells in the HSCs

major breakthrough of till & mcculloch

a single hematopoetic cell could self-renew and generate multiple differentiated cell types

stem cell factor

maintains and activates stem cells

erythropoeitin

makes more red blood cells

thrombopoeitin

makes more platelets

granulocyte-colony stimulating factor (G-CSF)

stimulates granulocytes, used clinically for HSC mobilization

monocyte-colony stimulating factor (M-CSF)

stimulates monocytes/macrophages

interleukins (ILs)

direct lymphoid differentiation

where are the sites of hematopoesis during gestation in order

yolk sac → liver → bone marrow, spleen is minor one

how can HSCs be identified?

NOT morphologically, surface markers such as CD34, c-kit, and Sca-1

flow cytometry

uses markers to isolate and quantify stem cells

as cells differentiate, they lose _ markers, and gain _ markers

stemness, lineage

lineage markers for lymphoid cells

CD19 and CD3

lineage markers for myeloid cells

CD13, CD33, CD123

lineage markers for macrophages

CD163

endosteal region/niche

signals to HSCs for quiescence, survival, and self-renewal, but these HSCs can become active and mobilize toward circulation when there’s a demand, has lots of signals to decrease proliferation and increase CDK inhibition

perivascular niche

signals to regulate proliferation and differentiation, has CXC12 signal that keeps stem cells in place and SCF that activates proliferation and differentiation

sinusoids

provide a route for HSC mobilization into and homing bloodstream

maintenance

move towards endosteal, signals for survival, quiescence, and self-renewal

mobilization

move towards perivascular, signals alter niche interactions allowing HSCs to move towards sinusoids and enter blood

homing

circulating HSCs can return to marrow and re-establish hematopoeisis

extramedullary sites

additional HSCs site like the spleen that can be used during stress, disease, or high demand

myelodysplastic syndromes

defective stem cells will create abnormal blood cell development, can affect RBCs (anemia), WBCs (infections), or platelets (thrombocytopenia/bleeding/bruising)

leukemias

abnormal proliferation and/or differentiation of hematopoeitic cells

acute leukemias

rapid increase in immature blast cells

chronic leukemias

slow accumulation of more mature but abnormal cells

cross-talk

bone marrow niche will cause reduction of osteoblasts and increase of osteoclasts, and this will reinforce the problems

treatment for leukemia and MDS

give someone else’s stem cells

mobilization drugs

G-CSF or CXCR4 blockers, will mobilize stem cells for collection through apheresis (blood collection)

how many stem cells do want to collect?

2 × 10^6 CD34 cells/kg of recipient weight

graft failure

stem cells did not repopulate

graft vs host disease

donor’s lymphocytes can kill you

parkinson’s disease

loss of dopaminergic neurons that results in uncontrollable movements as well as nonmotor symptoms

nonmotor symptoms of PD do not

show much recovery in treatment

early onset parkinson’s

ages 21-49 often linked to mutations of certain genes

late onset of parkinson’s disease

ages 50 and above, no single known cause

where are the dopaminergic neurons?

the substantia nigra pars compacta

levidopa

precursor to dopamine that is short term treatment to parkinson’s, patients will stop responding to it over time, administered with carbidopa to prevent premature breakdown in body

where will you transplant cells in PD?

the dorsal striatum, because if they are transplanted into the pars compacta then they will not know where to grow towards, the correct site will make the space between pre and post synaptic neurons shorter

pars compacta parts

dorsal tier that expresses calbindin, ventral tier that does not

calbindin

binds to Ca2+ to prevent too much that will kill neurons