Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer

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been developed. Such terms as competitive repopulation [88], repopulating units (RUs) [55], and competitive repopulating units (CRUs) [126] have been used to express stem cell activity quantitatively. RUs indicate the amount of repopulating activity and CRUs the actual number of stem cells. Thus these two measurements complement one another. When both values are determined for an experiment the mean activity of stem cells (MAS) can be calculated (MAS = RU/CRU). Ema and coworkers demonstrated a great diversity of repopulating activity in HSCs using these tools [37]. While all myeloid, B-lymphoid, and T-lymphoid lineages were reconstituted, the degrees of reconstitution in each lineage varied. They determined that the proliferation capacity of HSCs is dis-

sociated from the multilineage differentiation capacity and that this heterogeneity of HSCs is likely to result from their different levels of self-renewal capacity. Measurement of CRUs given by single HSCs suggested that the greater the self-renewal capacity the higher the repopulating activity. Similarly, HSCs with low RU regenerate themselves less than those with high RU. The heterogeneity in self-renewal capacity that these investigators observed implied that HSC self-renewal is not an unlimited capability [37].

These data may explain why bone marrow cells cannot be serially transplanted in mice more than four to six times, and support the generation-age model [111]. Allsopp and colleagues postulated that

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cell surface and cytoplasmic markers that are derived from both parental cells. However, fusion as a major contributor to the observed donor-derived chimerism has been excluded in several recently published studies in various organs [3, 54, 64, 120]. An even more controversial interpretation of this is the possibility of so-called therapeutic cell fusion [22, 23].

While our studies and others support the hypothesis that HSCs can adopt a tissue-specific phenotype by transdifferentiation, this view remains controversial. Others believe that the process really involves fusion and not transdifferentiation [132, 135]. Campbell and coworkers documented polyploidization of vascular smooth muscle cells in response to mechanical and humoral stimuli [24]. Thus, in some settings cell fusion can account for part of the accumulation of bone marrow-derived cells in the repair of injured tissue including vascular lesions. However, we have been unable to detect polyploidy in HSCderived cells within vascular lesions of the retina, choroid or iris following injury. While fusion may represent a significant component of the HSC repair observed in the liver and in vascular smooth muscle in some vascular beds, we consider it a rare event in the vasculature of the eye.

4.4 The HSC Niche

Stem cells are thought to occupy discrete spaces consisting of cellular elements, matrix, signaling molecules and pathways, collectively called a niche (Fig. 4.2) [90, 118]. The precise nature of these cellular and molecular components (the microenvironment) is thought to maintain the typical features of stem cells, i.e., quiescence, maintenance or expansion. Interaction of HSCs with their niche is critical for adult hematopoiesis in the bone marrow. HSCs must maintain the balance between quiescence and self-renewal in the stem cell niche as well as maintain long-term hematopoiesis.

The HSC niche comprises the endosteal surface of the bone marrow cavity, with osteoblasts being the most important cellular elements in maintaining HSCs [48, 81, 147]. Various signaling pathways and soluble molecules, including Wnt, Notch, transforming growth factor /bone morphogenic protein and Hedgehog, are known to be involved in all stem cell niches [42]. Of these, Wnt signaling appears to be most important for HSC self-renewal [109].

HSCs can be removed from the bone marrow of a donor and successfully engrafted by infusion into peripheral blood. Engraftment requires the transmigration of circulating HSCs from peripheral blood into the recipient’s niche. This engraftment depends upon the physical availability of niche space, as well

as homing signals. The spatial requirement is usually met by either chemical or radiological bone marrow ablation. The homing signals that induce this transmigration are poorly understood and only now beginning to be studied. It is thought that some of the same signals that induce HSC mobilization from the bone marrow are also involved in repopulating the HSC niche [1, 142].

4.4.1Molecular Mechanisms for Maintenance in the Niche

The Tie2-angiopoietin system appears to play a critical role in regulating proliferation, adhesion and survival of HSCs. Tie2+ HSCs have a higher long-term reconstitution activity than do Tie2 HSCs. Arai and coworkers put forth the idea that localization of Tie2+ HSCs on the bone surface is regulated by stem- cell-specific adhesion molecules such as N-cadherin [6]. Once the HSCs localize to osteoblasts, angiopoie- tin-1 (Ang-1) produced by osteoblasts may activate its receptor, Tie2, on HSCs and promote tight adhesion of HSCs in the niche, resulting in quiescence and enhanced survival of HSCs.

Tie2/Ang-1 signaling induces adhesion of HSCs to fibronectin and collagen [116, 128]. Following Ang-1 binding, phosphorylation of Tie2 results in activation of the phosphatidylinositol 3-kinase (PI3-K)/ Akt signaling pathway to promote endothelial cell survival [68]. Ang-1 leads to morphologic changes in HSCs in a 1-integrin-dependent manner and increases adherence of HSCs to the bone surface in vivo [7, 68]. The tight cell attachment of HSCs to stromal cells likely affects the cell-cycle status of HSCs. Since integrins trigger signaling that promotes cell survival, cell adhesion enhanced by Tie2/ Ang-1 signaling may protect HSCs from stress in combination with PI3-kinase/Akt signaling. Ang-1 administration provides clinical benefits that protect HSCs from anticancer therapy [25]. Thus Ang-1 maintains the in vivo repopulating ability of HSCs by inhibiting cell division and promotes quiescence of HSCs in vivo and Tie2 signaling keeps HSCs in the niche [7]. While Tie2 is required for postnatal bone marrow hematopoiesis it is not required for embryonic hematopoiesis. Tie2 is critical for the maintenance and survival of HSCs in the adult bone marrow and Tie2-deficient or kinase-deficient Tie2 cells are unable to occupy the adult bone marrow niche when competing with wild-type cells [7].

4.5 HSC Mobilization

Adult HSCs expand and differentiate exclusively in the bone marrow, from which they can be mobilized into the bloodstream. Cell-cell and cell-matrix inter-

4 Hematopoietic Stem Cells in Vascular Development and Ocular Neovascularization

Fig. 4.2. Hematopoietic stem cells can be induced to leave the stem cell niche, differentiate, and participate in neovascularization. This simplified schematic depicts some of the more well-known molecules and events involved in HSC mobilization. The lower half of the figure shows a damaged retinal microvessel, which releases several key molecules including SDF-1, VEGF and IL-8. These molecules act as mobilization signals, reaching the bone marrow stroma via the circulation, where they interact with numerous components of the HSC niche (top half of the figure). SDF-1 and IL-8 are both involved in G-CSF-mediated release and differentiation of HSC. SDF-1 and VEGF both act on osteoclasts, inducing their release of membranebound SCF. VEGF also acts directly to increase HSC proliferation, and may possibly influence their differentiation. SDF-1 mediates the release of MMP-9 from osteoclasts. Free MMP-9 can then cleave kit ligand, which influences HSCs, and MMP-9 can induce the release of HSCs from the stroma by impacting cell adhesion. Together these events result in the proliferation of HSCs and their differentiation into first multipotent progenitors and then into endothelial progenitors. The precise temporal sequence of differentiation and concomitant changes in

surface marker expression remain to be fully described, but it is likely that by the time mobilized cells translocate from the niche into the circulation they are fully committed to a specific terminal differentiation pathway. These circulating EPCs are then induced to leave the circulation by locally high concentrations of mobilization factors (among other, less clear signals) where they then differentiate fully and participate in reparative or regenerative processes. Here they are shown integrating with resident endothelium to form a compensatory collateral vessel

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actions are crucial to the proliferation and differentiation of HSCs in the bone marrow niche. Depending on the circumstances, HSCs can traffic from the marrow into the circulation in large numbers, a process termed mobilization [26]. Mobilization of HSCs is mediated by a complex interplay of changes in inte- grin-mediated adhesion as well as chemokine and growth factor receptor signaling.

Exogenous administration of granulocyte colony stimulating factor (G-CSF) is the standard method of inducing stem and progenitor cell release into the circulation in a clinical setting [26]. The chemokines interleukin 8 (IL-8) [112] and stromal-derived factor 1 (SDF-1) [56] both participate in G-CSF-mediated stem cell mobilization. SDF-1, as well as VEGF and PlGF (vascular endothelial growth factor and placen-

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tal growth factor, respectively), induces osteoclasts to secrete the metalloprotease MMP-9, whose action results in the shedding of the membrane-bound cytokine stem cell factor (SCF) from the bone marrow, releasing it into the circulation. Inhibiting SDF- 1 has been shown to reduce the degree of stem cell involvement in induced neovascularization [119].

4.5.1 The SDF-1/CXCR4 Axis

Endothelial cells in developing vascular beds express the SDF-1 receptor CXCR4, and animals deficient in either SDF-1 or CXCR4 do not form large vessels within the gastrointestinal tract, clearly indicating a role for this chemokine ligand/receptor in vascular development [82, 95, 127, 148]. CD34+ cells express functional CXCR4 and migrate in response to SDF-1 [104]. CXCR4 expression by endothelial cells of various origins is also well documented [93]. In endothelial cells, CXCR4 expression is increased after treatment with VEGF or basic fibroblast growth factor (FGF2) [115]. SDF-1 has also been shown to stimulate VEGF expression in a number of cells [67]. Any tissue production of SDF-1 results in mobilization of hematopoietic progenitors from the marrow to that tissue [105].

4.6Surface Marker Expression – HSC Identification

In the adult, HSCs can be distinguished from mature blood cells by their lack of lineage-specific markers and the presence of certain other cell-surface antigens [141]. Human cells express CD133 (prominin-1, a pentaspan membrane protein) [86] while murine HSCs express the cell surface molecules c-kit (CD117) and stem cell antigen-1 (Sca-1; Ly-6 A/E), and lack markers of differentiated peripheral blood cells (lineage markers, or Lin).

Most investigative studies have been performed on mice. CD117 is a receptor tyrosine kinase that is also expressed on mast cells, germ cells, and melanocytes. CD117 mutant mice have defects in HSC activity, lack pigmentation, and are sterile [15]. Mice deficient in c-kit have macrocytic anemia, decreased megakaryocyte numbers, decreased mast cell numbers, and defects in maintenance of B- and T-cell compartments [34, 137, 138]. CD117 appears to play a key role in the establishment of HSCs in the bone marrow “niche.” Blocking c-kit function in progenitor cells prior to transplant allowed normal homing to the marrow but defects occurred in lodging in the niche [33].

Sca-1 is a cell surface protein found not only on hematopoietic but also on mammary gland, cardiac, and mesenchymal stem cells in the mouse [11, 78,

124, 139]. Sca-1 is necessary for normal HSC activity as Sca-1–/– mice have defects in short-term competitive transplantation and serial transplantation [63]. In addition, Sca-1 knockout cells form fewer CFUs upon transplantation [63]. Because of these transplantation defects, it is believed that Sca-1 plays a role in HSC self-renewal.

Sca-1 belongs to a family of proteins bearing a UPAR (urokinase plasminogen activator receptor) domain. The UPAR domain is about 90 amino acids long and is important in cell adhesion and migration, modulates integrin function and regulates degradation of the extracellular matrix [18, 108]. Furthermore, UPAR directly regulates integrin expression and function by signaling and/or binding to integrins [18]. Therefore, Sca-1 is believed to modulate integrin function in HSC.

In addition to modulating HSC behavior, Sca-1 suppresses T-cell proliferation and alters signaling through the T-cell receptor (TCR) [125]. Sca-1 downregulation is required for T-cell differentiation [13]. Sca-1–/– mice have decreased megakaryocyte and platelet formation [63]. Interestingly, Bradfute and coworkers recently showed that Sca-1 affects CD117 expression and lineage fate of peripheral blood cells after transplantation and human CD34+ cell activity, but is not critical for self-renewal [19].

Although Sca-1 is routinely used to isolate many stem cells in the mouse, its biological role in HSC function is still only partially characterized. For example, such questions as how Sca-1 affects HSC engraftment and proliferation and how overexpression of Sca-1 affects HSC function remain unanswered.

4.7Surface Marker Expression – HSC Isolation

Isolation of mouse HSCs is a relatively straightforward, although time-consuming, process. The basic principle involves identifying specific cell populations with antibodies conjugated to either magnetic particles or fluorochromes, and then sorting the cells thus identified using magnetic or fluorescence activated cell sorting (MACS or FACS), respectively. HSCs and their progeny can be identified immunologically through their expression of distinct surface markers, the composition of which changes during differentiation. Typically some surface markers appear while others disappear (Fig. 4.1). Sca-1 and CD117 are most often used to distinguish HSCs from other cell types. These two markers yield a population of cells that is 95 % HSCs. Additional purification may be accomplished with considerable effort by first depleting lineage cells using antibodies to B220 (B-lymphocytes), CD3, CD4 and CD8 (T-lym-

Fig. 4.3. The generation of chimeric animals is depicted in schematic form. The bone marrow of recipient C57 BL/ 6J mice is depleted with lethal irradiation. At the same time, sorted HSCs from gfp+/+ donor mice are transplanted into these recipients. After 1 month, stable reconstitution is established through flow cytometry. These chimeric mice are then used for subsequent experiments. With the transplantation technique, the end functionality of the donor cells can be determined based on the presence of the gfp label in the cells

phocytes), CD11b (macrophages), Gr-1 (granulocytes), and TER119 (erythroblasts). This approach first uses magnetic bead-conjugated antibodies to deplete the lineage cells, followed by FACS using fluorescent-conjugated antibodies to CD117 and Sca-1 of the remaining cell fraction. For general reconstitution of bone marrow-ablated recipient animals, the use of FACS (CD177 and Sca-1) is usually sufficient. The following protocol is used routinely in our laboratory.

4.8 Methodologies

4.8.1 Extraction of HSCs from Donor Mice

1.Use 10to 20-week-old donor mice. TgN(GFPU)5Nagy (Jackson Laboratories, Bar Harbor, ME), bred in-house for homozygosity at the gfp locus, are used if tagged HSCs are needed. If HSCs are being used to reconstitute irradiated recipient mice, one donor mouse is needed for every ten recipients (Fig. 4.3).

2.Put to death donor mice; dissect away both hind legs and place on ice.

3.Working within a sterile field such as a laminar flow hood, dislocate the knee joint and cut through the joint to separate the thigh from the calf.

4.Dissect soft tissue away from the femur and tibia and slice off the joints, exposing the bone marrow.

5.Expel the marrow using a tuberculin syringe with ~0.5 – 1 ml sterile PBS (phosphate buffered saline) for each bone into a sterile centrifuge tube.

6.After collecting all of the marrow, homogenize by repeated pipetting.

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7.Centrifuge 300×g/10 min/4 °C; decant and resuspend in 2 ml of sterile PBS.

8.Aliquot into four tubes as follows:

a)500 μl sterile PBS, 30 μl BM cells.

b)500 μl sterile PBS, 30 μl BM cells, 1 μl phycoerythrin (PE)-conjugated rat anti-mouse Sca-1 (BD Pharmingen, San Jose, CA).

c)500 μl sterile PBS, 30 μl BM cells, 1 μl allophycocyanin (APC)-conjugated rat anti-mouse CD117 (BD Pharmingen).

d)To the remaining BM cells add 120 μl of PE-anti- Sca-1 and 120 μl APC-anti-CD117.

9.Incubate 15 min/4 °C.

10.Centrifuge 300×g/10 min/4 °C; decant and resuspend pellets in PBS (1 ml for tubes a, b and c, 10 ml for tube d).

11.Strain through filter tubes (cell-strainer cap, e.g., Falcon 2235) to remove large cell clumps and other debris.

12.Sort HSCs in tube d using a FACS Vantage SE Turbosort (BD Biosciences, San Jose, CA) or similar device to collect the fraction of cells positive for both CD117 and Sca-1; tubes a, b and c are used for calibration. Collect HSCs into sterile PBS with 30 % (v/v) serum during sort.

13.Combine sorted HSC fractions and wash by centrifugation 300×g/10 min/4 °C.

4.8.2Reconstitution of Bone Marrow-Ablated Recipient Mice

1.Recipient animals: 10 – 16 week old C57BL6/J.

2.Irradiate mice (850 – 900 rad) to ablate bone marrow.

3.Introduce ~5,000 HSCs into the systemic circulation of each recipient animal, either by tail vein or retro-orbital sinus injection.

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4.Maintain animals on antibacterial water (Bactrim or Baytril) for at least 2 weeks.

5.Assess bone marrow reconstitution after at least 1 month. A successful reconstitution will result in a high percentage of gfp+ leukocytes. Using flow cytometry analysis after a tail bleed, animals with at least 25 % gfp+ peripheral blood cells are considered reconstituted.

4.8.3 EPC Culture

Cell lines have been utilized to study many aspects of stem cell behavior. Stromal cell lines have been generated from the AGM region to study the molecular events involved in hematopoiesis and HSC differentiation within the AGM microenvironment [97]. These lines successfully maintained cultures of murine fetal liver-derived [97], marrow-derived [144], and human cord blood-derived cells [73]. Much focus has been given to characterizing growth factors believed responsible for the maintenance of hematopoiesis and differentiation in vitro [99].

Soluble factors are used for the preparation of medium required for the maintenance or expansion of stem cells in vitro. Commonly added factors are TPO, Flk2-ligand (FL), SCF, IL-6, G-CSF, and IL-3. Although some media have met with a degree of success, most investigators reported only a moderate expansion of stem cells in culture. It is difficult to maintain stem cells in culture without the presence of a supportive feeder layer of cells. Whether stem cells need to be in contact with these stromal cells is still a matter of debate. Both the AGM-S3 and DAS104 – 4 cell lines were incapable of maintaining early progenitors from fetal liver [97] or cord blood CD34+ cells [144] when they were not in direct contact with the stromal cells during culture. Thus stromal cells may be necessary for the maintenance of bone marrow HSCs because they provide anchorage as well as growth factors [98, 101]. As discussed below secreted molecules are equally important for the maintenance of stem cells in non-contact culture.

In our laboratory, EPCs are cultured using the endocult liquid medium kit (Stem Cell Technologies, Vancouver, CA) per manufacturer’s protocol. Briefly, 16 ml of human peripheral blood is collected into a CPT tube (BD Biosciences, San Jose, CA) and spun to obtain the mononuclear cell fraction (MNC). The MNCs are washed twice with PBS/2 % FBS and the pellet resuspended in 3 ml of endocult medium. The cells are plated onto a six-well fibronectin-coated dish (BD Biosciences, San Jose, CA) and kept at 37 °C, 5 % CO2 for 2 days. After 2 days, the non-adherent EPCs are harvested and further cultured for an additional 3 days to allow formation of endothelial colonies. Colonies are defined as a central core of

“round” cells with elongated “sprouting” cells at the periphery and are classified as early outgrowth colony forming unit-endothelial cells or CFU-ECs.

CD34+ cells are isolated from peripheral human blood using EasySep magnet kit (Stem Cell Technologies) per manufacturer’s protocol. Briefly 2×108 MNCs/ml are isolated from peripheral blood using CPT tubes. EasySep positive selection cocktail is added (100 μl/ml cells) to the MNCs and incubated at room temperature for 15 min. Following the incubation, 50 μl/ml of EasySep magnetic nanoparticles are added to the MNCs and incubated at room temperature for 10 min. The cells are washed five times within the magnet. The positively selected cells are then removed from the magnet and kept in HPGM media containing stem cell factor (25 ng/ml), thrombopoietin (50 ng/ml) and flk/flt ligand (50 ng/ml, R & D Systems, Minneapolis, MN). These factors help to keep the cells in the undifferentiated state.

CD34+ cells were obtained from the peripheral blood of healthy subjects using flow cytometry, labeled via PKH67 dye (Sigma, St. Louis, MO) and injected into postnatal day 1 mouse pups intravitreally. The pups were then subjected to the oxygeninduced retinopathy model as described by Smith [122]. The eyes were then removed, dissected, and examined for the presence of the green cells colocalizing with the vessels (Fig. 4.4).

4.8.3.1 Supportive Cells and Soluble Factors

Several different stromal cell lines have been reported to support HSC maintenance in culture. Oostendorp and colleagues compared gene expression profiles of two HSC-supportive and four HSC- non-supportive cell lines and focused on genes that might be involved in the common mechanism of HSC maintenance [100]. Thirty-one genes were found to be differentially expressed more than twofold in the HSC-supportive stromal cell lines compared with non-supportive lines. One-third of the genes expressed at a higher level in HSC-supporting stromal cells are secretory proteins. The authors favored the view that maintenance of HSCs is not supported by alternatives to VCAM-1-dependent adhesion pathways but by mechanisms dependent on soluble molecules. This favors the view that HSC maintenance on embryonic AGM-derived stromal cells is supported by contact-independent mechanisms [100].

The HSC-supportive cell line AFT024 expresses high levels of pleiotrophin (PTN), thrombospondin- 2 (TSP2) and insulin-like growth factor binding pro- tein-3 (IGFBP-3) [53]. PTN is a heparin-binding cytokine that in the embryo is expressed in metanephric and developing liver mesenchymal cells [9].

PTN is known as a guidance molecule for neurites and osteoblasts, probably by using surface-expressed syndecan 3 [70]. Whether PTN deficiency also affects HSCs has not been determined. PTN binds to several cell-surface molecules besides syndecan 3, including anaplastic lymphoma kinase [5], protein tyrosine phosphatase receptor Z (also known as RPTP-) [83], and cytoplasmic nucleolin [129]. Expression of the multifunctional cytoplasm-nucleolus shuttling protein nucleolin is observed in HSCs and is able to import endocytosed PTN into the nucleus [107].

IGFBP-3 was also highly expressed on AFT024 and the long-term culture-supportive cell line HS27a [49]. Our laboratory has extensively studied IGFBP-3 function in cultured endothelial cells. However, the function of IGFBP-3 in HSCs is much less clear. IGFBP-3 is found at high levels in serum, where it forms a heterotrimeric complex with IGF and the acid-labile subunit. IGFBP-3 was differentially expressed between HSC-supportive and non-sup- portive cell lines. These findings suggest that IGFBP- 3 could be one of the molecules commonly involved in the regulation of HSC behavior. Although IGFBP-3

binds IGF and modulates its availability, IGFBP-3 generates IGF-independent signaling. IGFBP-3 signals through an IGF receptor-independent pathway to phosphorylate Smad-2 and -3 and downregulate Smad-4 [38], which are involved in signaling through TGF family members. The responsible IGFBP-3 receptor, however, has not yet been identified. IGFBP-3 contains a nuclear localization sequence and IGFBP-3 binds intranuclear target genes, including p53, and retinoic acid receptor [76]. Thus, IGFBP-3 may affect HSC maintenance not only in its secreted form but also through its nuclear counterpart.

Other factors overexpressed in HSC-supportive cell lines include cathepsin K, FGF-7 (fibroblast growth factor), and pentaxin-related gene. Cathepsin K is a cysteine protease that is normally expressed by osteoclasts and that has been shown to play a role in bone resorption. One could postulate that the proteolytic activity of cathepsin K might be beneficial in the formation of the marrow niche.

After 7 days of culture on fibronectin, CD34+ mononuclear cells display an endothelial cell pheno-

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