Non-neural Stem Cells

characteristics of stem cells: “Stemness”:

—> self-renewal

—> can give rise to differentiating daughter cells, potency of stem cells impacts the cell types a stem cell can generate

—> maintenance of proliferative properties -> “nearly” unlimited proliferation

—> asymmetrical division: single cell asymmetry and population asymmetry

the terms “progenitor” and “precursor” are not clearly defined. They are used to describe the hierachy of a differentiation process from a stem cell to a differentiated cell. Since there are many “intermediate/transitory” cell types between a stem cell and a differentiated cell, it is important to define where/when a cell with it’s potency/function is present. Thus, progenitor and precursor are basically temporal definitions. A progenitor is a cell that a stem cell gives rise to. A precursor is a cell “upstream” of a differentiated cell.

Precursor cell - any ancestral cell type of a particular lineage

—> potency

• totipotent: embryo proper and extraembryonic structures

• pluripotent: embryo proper w/o extraembryonic structures

• multipotent: can generate all cell types of an organ/tissue

• oligopotent: can generate some cell types of an organ/tissue

• uni-/monopotent: can generate one cell type

—> resident tissue

• blood

• intestine

• brain

• lung

• hair follicle etc.

—> adult vs. embryonic (by origin)

• adult: tissue-resident: multipotent, oligopotent or monopotent

• embryonic: pluripotent (ICM or embryonic germ cells)

• induced pluripotent stem cells (iPSCs)

—> fast-cycling vs slow-cycling/quiscent

• fast proliferation: tissue homeostasis, replenishing tissues

• quiscence: maintenance of stem cell population

pluripotent stem cells can give rise to the entire embryo proper (not extraembryonic tissues). They exist in the inner cell mass of the early blastocyst and pluripotency is maintained by a special set of transcription factors that maintain pluripotency of the ESCs and prevent differentiation towards epiblast or primitive endoderm fates. The transcription factors are Oct4, Sox2 and Nanog. Oct4, Sox2, Klf4 and c-Myc are also the pioneer TFs that are used to induce pluripotency in iPSCs. (medium usually contains MAPK inhibitors -> proliferation inhibition)

How to generate them experimentally:

isolate them from ICM of early blastocyst (developemntal age of ICM determines whether they are naive or primed) and maintain in culture with pluripotency transcription factors. Or use embryonic germ cells (before differentiation into egg cell or sperm cell)

induce from somatic cells (iPSCs)

Tissues that undergo tissue homeostasis and replenish:

• blood

• intestine

• brain

• lung

• hair follicle

• epidermis

• etc

• multipotency (limited differentiation potential)

• can repair & maintain their resident tissues

• mostly epithelial cell types (except mesenchymal stem cell?)

• potency and stemness is maintained by specific niche architecture - self-renew but at lower rate than ESC

• tissue homeostasis

• tissue turnover

• repair upon injury

• staining for stem cell markers e.g. Lgr5 in intestine, Notch expression

• BrdU/Edu staining for quiescent and fast cycling stem cells:

  • proliferating cells take up marker

  • label-retaining —> quisecent

  • label-diluting —> active proliferation

mostly single cell asymmetry (asym. division):

• give rise to one differentiating daughter cell and one stem cell

or symmetric divisions:

• generates two stem cells and niche governs which differentiates, or two differentiatiated cells

• e. g. in intestinal crypts:

  • Lgr5+ SC divides and gives rise to two daughter stem cells

  • cell contact with Paneth cell maintains one as stem cell

  • the other differentiates, moves out of crypt and adapts transit-amplifying fate

specialized microenvironment that maintains stemness of stem cells

• stem cell

• progeny

• stromal and mesenchymal cells

• ECM -basal lamina (from blood vessels)

• long range cues (hormones or neuronal activity)

• niche cells

All components provide either adhesion signaling, contact-dependent signaling, autocrine singaling or paracrine signaling to maintain stemness

by paracrine signaling or contact-dependent signaling (mechanical cues from matix)

-spatial arrangement & composition of niche direct stem cell behaviour

• signaling can maintain stemness and regulate rate of proliferation:

  • if certain niche components are lost, the stem cell population can be lost

  • faster proliferation or differentiation (out of reach from niche factors)

  • loss of proliferation

Where do you find intestinal stem cells?

base of intestinal crypts

What are components of the intestinal stem cell niche?

• lgr5+ stem cells (CBCCs, fast cycling)

• 4/4+ position slow cycling stem cells

• paneth cells

• telocytes & pericryptal stromal cells - line basal surface of epithelium

• myofibroblasts

• smooth muscle cells

• transit amplifying cells (proliferative zone)

• ECM/ basal lamina

• differentiated cells (differentiation zone: matured epithelial cell types- Enterocytes, enteroendocrine cells,tuft cells, goblet cells)

How is proliferation and differentiation of intestinal stem cells regulated?

• proliferation of CBCCs via paracrine and autocrine signals from Paneth cells and stromal cells (Wnt, EGF, Notch)

• opposing morphogenic gradients Wnt2b+Rspondin&Noggin & BMP4

• division of CBCC at crypt base generates vertical displacement of cells —> shedding at vili tips (anoikis)

• daugher cells adapt secretory or absorptive lineage fates as they move out of crypt (loss of BMP inhibition via Noggin; Notch signaling regulates decision between secretory and absorptive fates)

Which signaling pathways are involved?

Where are the signals produced and which cells respond to them?

• Wnt & Rspondin:

  • produced by stromal cells (Wnt2b) and Paneth cells (Wnt3a), CBCCs respond, maintenance of proliferation

• BMP4

  • produced by stromal cells towards lumen, promotes differentiation of transit amplifying cells

• Noggin

  • produced by smooth muscle & fibroblasts in stroma at crypt base, represses differentiation of CBCCs

• EGF

  • /produced by stromal cells at crypt base) and Paneth cells, promote proliferation and survival of CBCCs

• Shh

  • secreted by stromal cells towards lumen, activates BMP secretion

• Notch

  • Dll4 expression by Paneth cells -> triggers NICD in CBCC ->maintenance

  • Notch ligand expression by TA cells: lineage decisions

• Ephrin

  • expressed in Niche, regulated positioning

How can you block WNT signaling originating from a particular source to study its function?

Inhibitors specific to signaling molecules ca isolate Wnt function

TCF/Lef TF KO

lipid modifications necessary for Wnt secretion:

flox enzyme that is essential for lipid modifications and put cre recombinase under control of cell type specific promoter e.g. Paneth cell specific promoter

Where do you find hematopoietic stem cells?

bone marrow

What are the components of the stem cell niche/s?

HSCs are arranged in two subniches that maintain a quiescent and a fast-cycling HSC population

—> endosteal niche (quiscent) and perivascular (fast-cycling) niche

• endosteal niche:

  long-term HSCs, progenitors, osteoblasts (secrete angiopoietin-1 and thrombopoietin that maintain quiscence), microvasculature, stromal cells

• perivascular niche:

  Short-term (active) HSCs, progenitors, blood vessels (perivascular HSCs are tightly associated with endothelial lining of vasculature), bone marrow-infiltrating axons, mesenchymal stem cells, stromal cells

How do hematopoietic stem cells home to their niche?

regulated via CXCL12/CXCR4 signaling and noradrenaline

• CXCL12 is secreted by CAR (CXCL12 abundant reticular cells) and MSCs

• acts via CXCR4 (expressed on HSCs) on HSCs and promotes proliferation and homing to niche

• BM-infiltrating axons secrete noradrenaline (circadian rhythm and stress-dependent) which acts on stromal cells and reduces CXCL12 secretion

• HSC enter circulation

Which signaling pathway are involved?

• angiopoietin-1 and thrombopoitein

• CXCR4/CXCL12

• c-Kit/SCF

• chemokine signaling

• neurotransmitter signaling (Noradrenaline)

• additional signaling factors: Wnt,TGFß,Notch/Jagged1, Integrins