Stem Cell: Niches, Mobilization and Homing 가톨릭대학교 안과학교실 김영훈

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

G-CSF mobilization model

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

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

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

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

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

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