Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this ‘missing self’ response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies, and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Abstract. SPARC (Secreted Protein Acidic and Richin Cysteine) is a Ca÷2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells.Bovine aortic endothelial cells, exposed to 2-40/zg SPARC/ml per 2 x 106 cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions.SPARC bound Ca ÷2 in both amino-and carboxylterminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca +2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca ÷2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.

SPARC (Secreted Protein, Acidic and Rich in Cysteine)/osteonectin is a secreted glycoprotein that exhibits restricted expression in murine adult and embryonic tissues and is associated with cell migration, matrix mineralization, steroid hormone production, cell cycle regulation, and angiogenesis. We produced a monoclonal antibody, MAb SSP2, against a Ca(2+)-binding region of SPARC and evaluated the immunoreactivity of normal and malignant tissue from 118 human samples. In normal tissue we found restricted and moderate reactivity with SSP2 in steroidogenic cells, chondrocytes, placental trophoblasts, vascular smooth muscle cells, and endothelial cells. Strong reactivity was found in fibrocytes and endothelial cells involved in tissue repair and in invasive malignant tumors, including those of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, ovary, and brain. We conclude that SSP2 is a useful reagent for detection of SPARC in human tissue. Given the broad reactivity of malignant tissues, we propose that SPARC expression might contribute to some aspects of tumor progression.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reichert's membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

of SPARC at 20 ,g/ml, 72% fewer cells were in S phase after a 24-hr period; a similar, but less marked, reduction was seen with peptide 2.1. Peptide 2.1 did not cause cell rounding, whereas peptide 1.1, a highly efficient inhibitor of endothelialcell spreading, exhibited essentially no activity with respect to cell-cycle progession. It therefore appears that the transient, inhibitory effect of SPARC on the entry of endothelial cells into S phase does not depend on the overt changes in cell shape mediated through cytoskeletal rearrangement.Regulatory signals affecting cellular growth and DNA synthesis operate on both positive and negative levels (1). Examples of the latter are the "tumor-suppressor" or "growth-suppressor" gene products, and the former includes polypeptide mitogens specific for different types of cells. In addition to the mitogenic growth factors, other proteins have been shown to play a role in stimulating cellular proliferation, such as the extracellular proteases thrombin (for review, see ref.2) and urokinase (3), the extracellular matrix (ECM) component fibronectin (4), and intracellular calmodulin (5).Negative control of mesenchymal-cell proliferation has also been described: a cell-surface fraction from confluent endothelial cells has been shown to inhibit endothelial-cell growth (6), and heparin prevents the progression of vascular smooth muscle cells through the G1 phase of the cell cycle (7,8).Mechanisms accounting for inhibitory effects on cell growth are largely unknown, although two pathways involving actin reorganization (9) and protein kinase C (7) have been experimentally confirmed. Studies in our laboratory have focused on the characterization of ECM proteins and their modulation of cellular behavior and phenotype. We initially identified a glycoprotein secreted by endothelial cells that were cultured at subconfluent density and exhibited high levels of proliferation and migration (10, 11). This "culture shock" protein was subsequently termed SPARC (secreted protein, acidic and rich in cysteine) by Mason et al. (12), who isolated the corresponding cDNA from parietal endoderm, a differentiated cellular population that is highly migratory but nonproliferating. SPARC is also identical to the proteins osteonectin (13,14) and and is highly conserved among several mammalian species.In the developing mouse, SPARC mRNA and protein have been localized to areas of active tissue morphogenesis (16,17). Expression of SPARC in the adult mouse appears confined to cellular populations that exhibit high rates of turnover (e.g., epithelium of the intestinal crypts), secretion (steroidogenic cells), and remodeling (e.g., lactating mammary gland) (16,17). Recent experiments have shown that SPARC effectively inhibits cell spreading (particularly that ofendothelial cells and fibroblasts) and binds to specific components of the connective tissue ECM in a Ca2+-dependent manner (18). Although the mechanism promoting changes in cell shape is not presently understood, we have proposed that interaction of SP...

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu 2؉ -binding sequence KHGK) exhibited a biphasic effect on [ 3 H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca 2؉ -binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu 2؉ -binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of iqjury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P < 0.02) transendothelial albumin flux in a dose-dependent manner at concentrations -0.5 jug/ml. At a fixed dose (15 pg/mi), exposure times > 1 h augmented (P < 0.005) albumin flux by 1.3-to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization ofF-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.The vascular endothelium presents a selective barrier that actively regulates movement of circulating macromolecules and cells into extravascular tissues and compartments (1, 2). Although mechanisms regulating this endothelial barrier are not well understood, a structure-function relationship appears to exist between endothelial cell (EC) shape and barrier function. Specific agonists can induce changes in EC shape coincident with the formation of intercellular gaps, which in turn provide a paracellular pathway for the flux of macromolecules (3-5). Actin organization is postulated to regulate EC shape as well as barrier function (5)(6)(7)(8)(9)(10)(11)(12). Actin filamentdisrupting agents increase endothelial permeability (6), prior stabilization of actin protects against increases in permeability (7-9), and mediators of permeability have been shown to induce cytoskeletal rearrangement (8-12 (20).Assay of Albumin Flux. EC were grown to confluence in 0.5 ml of medium on gelatin-impregnated polycarbonate filters (13-mm diameter, 0.4-,im pore size; Nucleopore) mounted in chemotactic chambers (ADAPS, Dedham, MA) as described (4). These chambers, which served as the upper compartment for the assay chambers, were inserted into wells of 24-well plates, each well containing 1.5 ml of medium and serving as the lower compartment of the assay chamber. 14C-BSA (Sigma; 30.1 pCi/mg of protein; 1 uCi = 37 kBq), the tracer molecule, was prepared in serum-free medium supplemented with BSA (34 mg/ml) to produce a final protein concentration equivalent to medium enriched with 10%o FBS. The baseline barrier function of each monolayer was established by application of an equivalent amount of 14C-BS...

S U M M A R Y SPARC (secreted protein, acidic, and rich in cysteine) is a matricellular protein that is present in the intervertebral disc; in man, levels of SPARC decrease with aging and degeneration. In this study, we asked whether targeted deletion of SPARC in the mouse influenced disc morphology. SPARC-null and wild-type (WT) mice were studied at 0.3-21 months of age. Radiologic examination of spines from 2-month-old SPARC-null mice revealed wedging, endplate calcification, and sclerosis, features absent in age-matched WT spines. Discs from 3-month-old SPARC-null mice had a greater number of annulus cells than those of WT animals (1884.6 Ϯ 397.9 [mean Ϯ SD] vs 1500.2 Ϯ 188.2, p ϭ 0.031). By 19 months discs from SPARC-null mice contained fewer cells than WT counterparts (1383.6 Ϯ 363.3 vs 1466.8 Ϯ 148.0, p ϭ 0.033). Histology of midsagittal spines showed herniations of lower lumbar discs of SPARC-null mice ages 14-19 months; in contrast, no herniations were seen in WT age-matched animals. Ultrastructural studies showed uniform collagen fibril diameters in the WT annulus, whereas in SPARC-null disc fibrils were of variable size with irregular margins. Consistent with the connective tissue deficits observed in other tissues of SPARC-null mice, our findings support a fundamental role for SPARC in the production, assembly, or maintenance of the disc extracellular matrix.