stem cell exam 2

what typically happens when u inject SCs

disappear rapidly, but have an effect

tie off femoral artery

if left alone, it will degenerate

if ASC (adipose stem cell) is injected, there is improvement in survival

ASC characteristics

source of beneficial factors

do not differentiated with marked frequency

many uses once separated

• Elizer sheet with antibodies to proteins, take mixture of cell medium, black spot identify which molecules are present

• mainly heart

First Direct Demonstration In Vivo of the Paracrine Principal in 2007

attempt to identify factors present

• If u knock out hgf (hepatocyte growth factor), you drop the efficacy of the condition medium that contains a ton of factors

  • It does not completely wipe out, so there is more than one factor that impacts this

HGF (hepatocyte growth factor) produced at high levels by ASCs

• potent angiogenic factor

• inhibits apoptosis (promotes cell survival

• motogenic (scatter factor - stimulates cell movement)

• ligand for c-Met receptor

where do adipose stem cells surround

small blood vessels; they are the repair stations of the body placed on the highways of the body. they know to go here via Pdgf receptor beta

Pdgf receptor beta

cells drape around vasculature to put their growth factors into the blood circulation

ASCs can help with…?

stroke, retinopathy, emphysema/ALI, diabetes, heart attack/failure, erectile dysfunction, limb salvage (threatened amputation), wound healing, peripheral neuropathy, multiple sclerosis, alzheimers

secretomes can help with…?

stroke, ALI, transplanted pancreatic islet stabilization, leg salvage, ALS, kidney failure, pancreatitis, wound healing

getting concentrated asc-cm

culture cells, harvest medium and remove cells and debris, concentrate

rat model of stroke/ cerebral palsy due to hypoxia

Tie off carotid artery for a short amount of time.

Cerebral cortex dies . put asc media back in and u recover from the stroke.

• asc secretes substances that protect brain from injury

• This has to be done fast though

exosome

also known as secretomes. it is just medium

cell membrane blobs, so they are lipid based

contain growth factors

• in capsule to prevent breakdown in the blood

• put into any damaged place in the body

impacts of secretome

reduce stroke volume, functional impairment, and death

doubles survival time in ALS mice

reduces inflammation and preserved barrier function in acute lung injury

increase: angiogenesis and neurogenesis

decreases: apoptosis, inflammation (macrophage infiltration, microglia activation, and t-lymphocyte activation)

secretome vs asc

since secretome is j medium, they can be more easily stored and sustained (cells u have to keep alive, which is hard)

u also dont need to have secretomes derived from yourself

enhancement of organ survival for transplant

many viable hearts are not able to be used due to ischemia

put heart into medium with secretomes, max transport time doubles from 2 to 4 hours

improves heart recovery

cell to secretome therapy

cells - autologous (pt own cells), non-expanded (cells not manipulated in lab to be cultured)

secretomes - allogenic, expanded

asc conclusion

found in stromal cells of adipose tissue. readily available

grow into blood vessels

promote survival and tissue rescue

tissue repair cells located on vessels throughout the body

• active in many diseases

asc secretome can address

limits brain injury in stroke and cerebral palsy, behaves dose-dependently

peripheral vascular disease and acute lung injury/barrier leak

acute renal injury

acture pancreatitis

increase organ storage time and recovery

What do hematopoietic stem cells do?

Make all the different types of blood cell types

at least 8 different lineages

Where do hematopoietic stem cells arise from

Single progenitor from bone marrow

hsc

Undifferentiated founder cell giving rise to all blood cell lineages. Capable of self renewal

Multipotent progenitor

Partially differentiated cell that gives rise to multiple lineages. Limited ability to self renew

Committed progenitor

Lineage restricted, but not fully differentiated

Hscs have what characteristic on their markers

Undifferentiated founder cell that doesnt express the markers for any of the individual lineages

thus you can take bone marrow and sort out what does have markers

Characteristics of hscs

Capable of self renewable

Asymmetric cell division - one differentiated daughter cell and one that remains a stem cell

characteristic of neonatal lymphocytes

not made new and live for the rest of the person’s life

why do some cells not last more than a day

restriction on lineage development

primitive hematopoiesis

yolk → liver → spleen → bone marrow

nucleated erythrocytes w/ embryonic/fetal hemoglobins.

potential differences in myloid and lymphoid development

• diff hemoglobins in fetus than adult

• diff gas exchange rates for each hemoglobin

• some markers only present in embryonic development stage

make nucleated rbcs and primitive macrophage (nurse cell)

yolk sac

blood islands here are first site of hematopoiesis, and is not in the organism itself

process is from outside to inside

definitive hematopoiesis

liver → bone marrow

occurs 2/3 through fetal development

enucleated rbcs producing b-globin. mature patterns of myeloid and lymphoid development

common link btwn sites of development and blood vessels

• aorta when places into radiated tissue can save it

hemangioblast

can develop epithelial and hematopoietic cells. precursor to blood and blood vessels

sites of embryonic hematopoiesis

aorta, gonads mesonephros: region of intraembryonic mesoderm where HSC are found

yolk sac: extraembryonic membrane. hemogenic endoderm promotes production here

fetal liver

bone marrow

Different stem cells that develop in each site or stem cell migration

-       During transition periods, we would see periods of picking up and falling

-       Isolate the circulating blood and putting them into the assay, the number of progenitors spikes (more in circulation) during the move.

decline of progenitors in the yolk sac is followed by rise in the liver. this suggests the migration of cells from one organ to another via the bloodstream

seeing how HSCs work

culture to see what they turn into

use all stages and see how they work in transplantation

HSC assays

one thing that is determinative of a HSC

• be able to transplant it into an irradiated mouse and have it recover

• ultimate test of what an HSC is

HSC can…

a single HSC can repopulate all hematopoietic lineages for the life of an individual

long term multi lineage repopulation - demonstrates stem cell function

movement of hematopoietic cells - can make transplants to show that they move (ie tibia bone marrow experiment)

HSC are found

in the agm region and has a common progenitor with the vascular system.

• This was thought to only be during fetal development, tube with vessels and stuff that bathed the blood. However they are still related

mesenchymal stem cell (MSC)

other bone marrow stem cell

can make: all blood and adipocytes, bone, osteoblasts, nervous system tissue, and muscle in culture

heterogenous

not serially transplantable (loses function/properties when injected into new host)

what happens when u put msc in damage

do not implant

works through paracrine factors and acts as an immune reset switch

direct immune modulation

engineer to express specific cytokines/proteins

source of transplant cells

msc therapies may result in

bone sarcomas

mscs in cartilage repair therapy

implant into porous ceramic implant to graft into knee to replace damaged cartilage

injecting mscs for heart tissue repair

adult stem cells regenerate damaged heart muscle

induce new blood vessel formation and proliferation of existing vasculature

msc cured graft vs host disease

derived from 75 people so too expensive to actually be a cure

immune reset seems to work in other diseases as well

msc future

hope for autoimmune diseases

maybe good for allo bmt (Allogeneic blood or marrow transplantation)

diabetes

dimer chances for neural diseases

6 aspects of the stem cell (IMPORTANT)

Defined by functional definition

Proliferation

Self renewal over time

Generation of large progeny (defining what large means is an issue)

Multilineage differentiation potential (give rise to all cell types from the tissue they are obtained)

Regenerate tissue after injury

Neural stem cells are defined by their behaviors

o This creates issues bc u have to do it retrospectively.

o Is their action normal or abnormal (when u force them to act)

Ie. put stem cell into medium, but conditions aren’t right, so it doesn’t act like one. u would then be forced to not call it a stem cell.

how much can a neural stem cell divide (how long do they last)

as much to fill 10 to the 30th olympic size swimming pools

10 to the 50th amount of cells after 52 passages

NSCs are found

in discrete regions of the rat brain. hot spots where continuous cell genesis occurs

• subventricular zone - migrate to olfactory orb where they form new functional tissue

• dentate gyrus in the hippocampus - cells migrate to diff part of the hippocampus involved in the retrieval of memory

in each region 9000 cells generated every day

single NSC can make what in culture

progeny of that single cell would differentiate into : neurons, astrocytes and oligodendrocytes

transplantation of NSCs

need to be purified to prevent adverse effects

• might all turn into astrocytes, reducing diamine which increases pain

  • inflammation and immune responses

neural cell genesis

Once the brain gets injured, the cells migrate and differentiate to try and repair this

Blocking cell genesis causes reduction in memory

Stimulating environment increases cell genesis

Exercise increases

Stress decreases

neurosphere

90% of cells lost the proliferating characteristics . most of the sphere is made from progenitor cells, not stem cells.

- NSCs only 0.1-0.3% of the cells in the neurosphere

• only small percentage forms new neurospheres

wnt2a on nsc

increase the number of DA neurons by inducing progenitor proliferation in ventral midbrain cultures

two places where esc are

basal lamina (epidermis) and bulge

epidermal proliferative unit

make a line. epidermis is full of this

a lot of stem cell concepts were formulated from these

confirmed w lacZ cells

esc in hair bulge

can isolate them

go through phases of death and reconstruction

bulge does not get destroyed

originally thought to be in the matrix

source of esc in development and in adult during wound healing

discovery of esc in bulge

label mouse and wait a couple weeks

• see where leftover cells are sitting

• stem cells are slow (in bulge)

• fast ones are transit amplifying

what is present in the bulge and also an hsc marker

cd34

have to be careful to choose a marker that doesn’t have commonality

where do bulge cells go

make whisker blue and transplant bulge.

go to the surface of the skin and then downwards beck into the base of the hair in the bulb

bulge cells generate all epithelial lineages of hair as well as sebaceous glands

esc wound healing

lac-z reporter cells from the bulge migrate into the wound.

if there are no bulge cells, the wound still heals

• they do what they do but arent essential

bulge cells come streaming out of the follicle, but disappear over several weeks.

• no longer bulge like and are now epidermal like

  • only in mus mice

• in spiny mouse, they can regenerate full thickness wounds with new hairs

  • retain bulge stemness and make new hairs

does the bulge generate epidermis normally

no, very small contribution (1%)

esc conclusions

true stem cells proliferate infrequently, but are perma clonogenic (make colonies)

make TA cells (proliferate highly and low clonogenicity)

• transit amplifying cells (stem cell → transit → differentiated)

bulge stem cells are important for hairs/sebaceous glands/ epidermis

lineage tracing of stem cells is important

markers can be rna sequenced and characterized (niche)

stem cells migrate

esc niche

col IV, laminin, integrin

keratins - 5 14 1 10

drivers of stratification

mitotic spindle orientation (parallel to perpendicular)

changes in gene expression (ie p63)

human epidermis

5 layers - corneum, granulosum, spinosum, germinativum

rete ridges - 2 levels of integrin at top and bottom

• high integrin cells proliferate extensively

• low proliferate less

hair follicles develop at the same time as stratification

pilosebaceous unit

outer root sheath

inner root sheath

matrix

dermal papilla

hair cycle

anagen (makes new follicle) → catagen (degeneration) → telogen (resting phase)

• retracting base of follicle stops at bulge in telogen

need wnt, bmp, shh, fgf, egf, nfkb, notch

hyperplasia

increase total number of fibersh

hypertrophy

increase size of each fiber

• born with a fixed number of fibers, so postnatal growth is mostly due to this

muscle regeneration

regenerated fibers have centrally located nuclei

• mature cells are normally on the outside where the satellite cell is

need multiple cell types to regenerate muscle: blood vessels and immune system

what are muscle stem cells called

satellite and FAP (fibro adipogenic progenitors) cells

are satellite stem cells really stem cells

give rise to diff cell type (unipotent) and are able to self-renew

transplanted sc retain their stemness

• repopulate host stem cell niche

essential for regeneration

loss of muscle mass and function

sc not involved in maintance of tissue w/o injury

failure of old/diseased muscle is due to decline in sc numbers and function

• cell intrinsic changes - aged sc less capable of niche repopulation and differentiation

• enviro changed - hormones, GF, metabolism, etc

parabiosis

connecting the blood circulation of two mice

young blood causes an old mouse to regen its muscle as efficiently as young mouse

dont know youth factor

rejuvenates other tissues such as nervous system, heart, and liver

anti inflammatory drugs

inhibit regeneration because they prevent inflammation

fap cells

pro-regenerative cues

maintenance of ecm

provide pro-myogenic factors

MuSC conclusions

skeletal muscle one of few tissues that can regenerate

satellite cells - dedicated sc population

• unipotent adult stem cells

muscle tissue loses regen capacity with age and disease

• intrinic and enviro functions

regen involves many diff cell types (i.e., regenerative macrophages)

• nsaids impact regen

muscle regen experiment

typical model is to inject snake venom

make sc blue with lacZ.

regenerated cells are all blue (know this bc nuclei is in the middle)

marker for sc

pax7. if u delete the wound does not heal

transplanted pax7 cells retain stemness