1. Stem Cell: Niches, Mobilization and Homing
가톨릭대학교 안과학교실 김영훈

2. Stem cell treatment basics
Niche
Mobilization
Trafficking and Homing
Mesenchymal stem cell

3. Stem cells as powerful therapeutic candidates
Lego block for regenerative medicine and tissue engineering
Integration into tissue
Molecular signals

4. Current stem cell therapy
Rationale
Replenish and maintain cell niche with exogenous SC or progenitor cells
Restore regenerative potential of tissue

5. Ex vivo-cultivated stem cells
Harvest resident SC and expand in cultures
Strategy
transplant back
seed into prefabricated 3-D scaffold and then implant back
cell-seeded scaffold incubated to create a tissue-like construct before transplantation
 SC exposed to biological, chemical, or physical stimuli that promote formation of appropriate repair tissue

6. Niche
in vivo milieu
subset of tissue cells and broad spectrum of extracellular substrates
regulates stem cell survival, self-renewal, and differentiation
stem cell niches present in many adult organs and tissues including brain, bone marrow, peripheral blood

7. BM: main reservoir for many types of SC
BM microenvironment critical in maintenance of SC
HSPCs preferentially localize to a perivascular localization or near endosteum
osteoblast lineage cells required to maintain ‘endosteal SC niche’
Pivotal role in maintenance of various niches and SC fitness in steady conditions
 Dynamic balance in which small numbers of SC constantly leave the BM, enter tissues, and travel back to the BM or peripheral tissue-specific niches

8. Key function of stem cell niches
maintain constant number of slowly dividing SC by balancing proportions of quiescent and activated cells
activated by normal need to maintain tissues, or by disease or tissue injury
different tissues have varying degrees of latent regenerative potential
 tissues with low regenerative capacity or age-related decline require external stimulation and/or SC to catalyze repair

9. Mobilization Increase in circulating HSPCs in response to
systemic or local inflammation
strenuous exercise
stress
tissue/organ injury
administration of agents
* HSPC: hematopoietic stem/progenitors cells

10. Basic mechanism of mobilization
release of proteolytic enzymes by myeloid cells in BM (eg. MMPs, elastase, cathepsin-G)
decreased activity of inhibitors of proteolysis (eg. serpins)
permeabilization of the BM-blood barrier

11. Mobilizing agents
Most important mobilizing agents currently employed clinically
cytokines (e.g., G-CSF),
cytostatics (e.g., cyclophosphamide)
CXCR4-blocking molecule (Plerixafor (AMD3100)
very late activation protein 4 (VLA-4)-blocking molecule: BIO4860
CXCL2 (growth-related oncogene protein-beta (Gro-β))
CCL3 (macrophage inflammatory protein-1alpha (MIP-1α)
CXCL8 (IL-8)
* receptors not expressed on HSPCs, effects mediated by BM-residing accessory cells
CXCR4 receptor agonists: most promising
SDF-1α peptide analog (CTCE-0021)
Pepducin (ATI-2341

12. Granulocytes and monocytes egress first
Secrete proteolytic enzymes that disintegrate endothelial barrier  permeabilize blood-BM barrier
‘Pave the way’ for HSPCs that follow

13. G-CSF Granulocyte colony-stimulating factor (G-CSF)
Most commonly used agent
G-CSF-mobilized hematopoietic stem/progenitors cells (HSPCs)
more rapid engraftment and superior overall survival in comparison to BM

14. G-CSF model of HSPC mobilization
Delayed, peak levels of circulating HSPCs achieved after 5–7 days of Tx
Broad spectrum of HSPCs mobilized
Myeloid, megakaryocytic and erythroid progenitors also
Mobilized HSPCs have characteristic phenotypic features
 distinct from HSPCs in BM under steady-state conditions

15. Mechanism of G-CSF mobilization
G-CSFR signaling required
G-CSF mobilizes HSPCs through a hematopoietic intermediate
G-CSF  hematopoietic intermediary  trans-acting signal(s)  HSPC mobilization
Osteoclasts may be the target cell

16. HSPC Regulation CXCR4/CXCL12 axis VCAM-1/VLA-4 axis
Urokinase-type plasminogen activator receptor (uPAR)
Complement
C-kit/KitL axis

17. CXCR4/CXCL12 axis CXCL12 (aka stromal-derived growth factor-1 (SDF-1)
Major receptor CXCR4
Crucial role in regulating HSPC trafficking, homing and maintenance
CXCR4 signaling
 provides key retention signal for HSPCs in BM
CXCL12 expressed at high levels in BM
 potent chemoattractant for HSPCs
Numerous pathways modulate mobilization either by altering baseline CXCL12 production or CXCR4 response

18. G-CSF and CXCL12/CXCR4 axis
Suppression of CXCL12/CXCR4 axis
 dominant mobilization mechanism by G-CSF
Early, transient increase in CXCL12 production
Prolonged treatment causes progressive decrease in CXCL12 mRNA
Lowest levels of CXCL12 mRNA or protein correlates with maximal mobilization
Also decreased CXCR4 expression on mobilized HSPC

19. G-CSF treatment and BM
osteoblast lineage cells: source of CXCL12 in BM
CXCL12 ↓↓↓
mature osteoblast number and function ↓↓↓
surviving osteoblasts reduce expression of CXCL12 mRNA by ½
osteoblast suppression is a common feature of HSPC mobilization by other cytokines, including Flt3 ligand and kitL

20. Neutrophil-derived proteases induced after G-CSF treatment
MMP-9, cathepsin G, neutrophil elastase
 proteolytic environment in BM
Cause mobilization by cleaving a variety of HSPC supporting molecules, such as kitL, VCAM-1, CXCL12 and CXCR4
Neutrophil proteases may augment G-CSF-induced HSPC mobilization in appropriate genetic backgrounds

21. VCAM-1/VLA-4 axis Vascular cell adhesion molecule 1 (VCAM-1)
Major ligand: very late antigen 4 (VLA-4, aka α4β1 integrin)
Anchor HSPCs to BM stromal cells and regulate HSPC trafficking between BM and peripheral sites
G-CSF induces proteases that cleave VCAM-1

22. Regulation by uPAR urokinase plasminogen activator (uPA, urokinase)
urokinase-type plasminogen activator receptor (uPAR)
uPAR cleaved from cell surface by plasmin with G-CSF treatment
Mechanism of HSPC mobilization
disrupt uPAR interaction with integrins
inhibit CXCR4 signaling by soluble uPAR fragments
induce HPSC migration by soluble uPAR fragments

23. Complement regulation
G-CSF may activate complement by via antibody activation
HSPCs express receptor for C3a
Augments chemotaxis to CXCL12
negatively regulate mobilization
C5 positively regulates mobilization
No C5a receptor: not direct effect
may be related to neutrophil activation
Membrane attack complex (MAC) formation leads to RBC lysis
Releases lipid sphingosine-1-phosphate (S1P)
Potent chemoattractant for HSPCs
Responsible for egress of HSPCs
Permeabilization of endothelial barrier in BM sinusoids triggered by activation of complement cascade

24. C-kit/KitL axis High levels of c-kit
kitL crucial role in promoting HSPC quiescence and self-renewal
G-CSF induces production of proteases that cleave c-kit and kitL, releasing both as soluble forms

25. HPSC egress from BM by CXCR4 signaling disruption
BM endothelial cells may provide second signal directing neutrophil migration from BM into circulation
‘Tug-of-war’ model
CXCL2 expression by endothelial cells (release) and CXCL12 expression by endosteal osteoblasts (retention)
 regulate neutrophil release from BM
S1P generated during G-CSF Tx by complement activation may contribute to HPSC migration into blood
 gradient

26. G-CSF mobilization model

27. Trafficking #1
Interactions with vascular endothelial cells via selectins, membrane-bound growth factors, and their ligands/receptors
Activation of integrin adhesiveness
Endothelial transmigration
Chemotaxis

28. Trafficking #2 Interstitial migration/ active amoeboid movement
Recognize and obey extravascular cues
Identification of primary navigational clues
Render SC surface more
responsive to homing factors or guidance cues

29. Homing
SC recruitment to injured tissues or their navigation to other target niches/locations following mobilization
SC ability to find its way to a particular anatomic destination, often via bloodstream
Homing required for all SC-based therapeutics, either endogenous or artificially administered
Neighboring healthy tissue may also provide stem/progenitor cells
Recruitment process may be facilitated by external stimuli, such as cell homing factors

30. Endogenous stem cell homing strategies
Retrieve initial healing capacity of a tissue by pharmacological means
Activation of endogenous stem cells from either blood or a tissue-specific niche in aged or diseased individuals
Reactivation of endogenous stem cell potential

32. Mesenchymal Stem Cells
stromal cells that possess capacity to self-renew and multilineage differentiation
isolated from a variety of tissues such as,
umbilical cord, endometrial polyps, menses blood, bone marrow, adipose tissue, etc
ease of harvest and quantity obtained
Differentiation controlled by
Regulatory genes, growth factors, induction chemicals
microenvironment built with biomaterial scaffolds

34. Take home message Niche
in vivo milieu containing various SC and supporting substances
regulates SC survival, self-renewal, and differentiation
BM main reservoir
Steps in SC treatment
Mobilization
Trafficking
Homing
Most commonly used agent for mobilization: G-CSF
Acts mainly by suppressing CXCL12/CXCR4 axis
Proteolytic environment in BM