SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.Generation of cardiovascular cells from human pluripotent stem cells (hPSCs) in culture could provide a powerful model system for investigating cellular interactions and molecular regulators that govern the specification, commitment and maturation of these lineages, as well as a unique and unlimited source of human cardiomyocytes for drug testing and regenerative medicine strategies [1][2][3][4] . Translating this potential into practice, however, will depend on the development of technologies that enable the reproducible generation of highly enriched populations of cardiomyocytes, as contaminating cell types could affect drug Reprints and permissions information is available online at

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“…Immunoprecipitation analyses revealed a band the size of that previously described for the SIRPA protein ( Supplementary Fig. 4b) 12 .…”

Section: Surface Of Markers Expressed On Hesc-derived Cardiomyocytessupporting

confidence: 63%

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

et al. 2011

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“…The ligand for SIRP␣ was recently found to be CD47, an integrin-associated protein with multiple functions in immunological and neuronal processes (10,11). Consistent with this observation, SIRP␣1 was shown to be phosphorylated in response to cell adhesion in macrophages as well as in nonhematopoietic cells (12,13).…”

supporting

confidence: 73%

Cutting Edge: Signal-Regulatory Protein β1 Is a DAP12-Associated Activating Receptor Expressed in Myeloid Cells

Dietrich¹,

Cella²,

Seiffert

et al. 2000

The Journal of Immunology

145

115

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Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRPβ1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRPβ1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRPβ1/DAP12 complex formation was required for efficient cell-surface expression of SIRPβ1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRPβ1 is a new DAP12-associated receptor involved in the activation of myeloid cells.

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“…SHPS-1 is implicated in the recruitment and activation of SHP-1 at sites near the plasma membrane in response to cytokines or integrin-mediated cell adhesion in macrophages. 12,19,20 The mutant form of SHPS-1 lacks most of the cytoplasmic region of the protein and is thus unable to bind SHP-1, 34,35 a protein tyrosine phosphatase that negatively regulates multiple functions of hematopoietic cells including macrophages. [21][22][23] It is thus likely that the enhanced phagocytic activity of macrophages from SHPS-1 mutant mice is attributable to both a loss of SHPS-1 expression and deletion of the cytoplasmic region.…”

Section: Discussionmentioning

confidence: 99%

“…7,9,[15][16][17][18] SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. In macrophages, tyrosine-phosphorylated SHPS-1 binds SHP-1, 12,19,20 which is implicated in negative regulation of the functions of a variety of hematopoietic cells. [21][22][23] The complex of SHPS-1 and SHP-1 is thus thought to regulate macrophage functions in a negative manner.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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SHPS-1 promotes the survival of circulating erythrocytes through inhibition of phagocytosis by splenic macrophages

Ishikawa-Sekigami

Kaneko

Okazawa

et al. 2006

Blood

109

112

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IntroductionThe lifespan of circulating red blood cells (RBCs) is approximately 120 and 40 days in humans and mice, respectively, and is determined by their production in bone marrow (BM) and their clearance from the peripheral circulation, predominantly in the spleen and liver. [1][2][3] The production of RBCs is controlled by the primary erythropoietic regulator erythropoietin, 2,4 whereas clearance of old RBCs by the spleen is achieved mostly as a result of their specific recognition and phagocytosis by splenic macrophages. 5,6 The precise molecular mechanism by which splenic macrophages recognize senescent RBCs for phagocytosis is largely unknown, however. [1][2][3] Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), 7,8 also known as signal-regulatory protein ␣, 9 brain immunoglobulin (Ig)-like molecule with tyrosinebased activation motifs, 10 and p84 neural adhesion molecule, 11 is a transmembrane protein that is especially abundant in macrophages. [12][13][14] The putative extracellular region of SHPS-1 comprises 3 Ig-like domains, and its cytoplasmic region contains 4 tyrosine phosphorylation sites that mediate the binding of Src homology 2 domain-containing protein tyrosine phosphatases designated SHP-1 and SHP-2. 7,9 Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors and cytokines as well as by integrinmediated cell adhesion to extracellular matrix proteins. 7,9,[15][16][17][18] SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. In macrophages, tyrosine-phosphorylated SHPS-1 binds SHP-1, 12,19,20 which is implicated in negative regulation of the functions of a variety of hematopoietic cells. [21][22][23] The complex of SHPS-1 and SHP-1 is thus thought to regulate macrophage functions in a negative manner.CD47 is a ligand for the extracellular region of SHPS-1. 24,25 This protein, which was originally identified in association with ␣v␤3 integrin, is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, 5 putative membrane-spanning segments, and a short cytoplasmic tail. 26 CD47 and SHPS-1 constitute a cell-cell communication system (the CD47-SHPS-1 system) that plays important roles in a variety of cellular processes including cell migration, 27,28 adhesion of B cells, 29 and T-cell activation. 14,30 In addition, the CD47-SHPS-1 system is implicated in negative regulation of phagocytosis by macrophages. CD47 is highly expressed on the surface of RBCs, where it associates with the Rh protein complex instead of with integrins. 31 The rate of clearance of CD47-deficient RBCs from the bloodstream was found to be markedly increased compared with that of wild-type (WT) cells. 6,32 Furthermore, the phagocytosis of CD47-deficient RBCs by splenic or BM-derived macrophages was greatly enhanced Supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer; a Grant-in-Aid for Scientific Research (B); a Grant-in-Aid for Young Scie...

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SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages

Cited by 101 publications

References 25 publications

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

Cutting Edge: Signal-Regulatory Protein β1 Is a DAP12-Associated Activating Receptor Expressed in Myeloid Cells

SHPS-1 promotes the survival of circulating erythrocytes through inhibition of phagocytosis by splenic macrophages

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