A carbohydrate-binding protein assayed by its ability to agglutinate formalinized sheep erythrocytes is synthesized between 3 and 9 hr after Dictyostelium discoideum cells are deprived of food, as the cells become cohesive. Agglutination of erythrocytes by this protein was inhibited by N-acetyl-D-galactosamine, D-galactose, and Lfucose, but other monosaccharides had little or no effect. The protein bound completely to Sepharose 4B, and was isolated in highly purified form by elution with D-galactose. It appears to be present on the surface of cohesive but not vegetative slime-mold cells. Previous work has demonstrated that a protein factor assayed by its activity in agglutinating formalinized sheep erythrocytes can be extracted in increasing amounts from D. discoideum cells as they differentiate after removal of food (3). In a 12-hr period over which the cells become progressively more cohesive, the specific activity of the agglutination factor in cell extracts increases by over 400-fold (3). In this report, we show that the appearance of the agglutination factor in D. discoideum is closely correlated with development of cell cohesiveness, as measured by a newly devised quantitative assay. We also show that the factor is a carbohydratebinding protein with a high degree of specificity. We present preliminary evidence that the factor is detectable on the surface of cohesive slime-mold cells. buffer consisting of 40 mM potassium phosphate buffer (pH 6.5) containing per liter 1.5 g of KCl, 0.61 g of MgSO4, and 0.5 g of streptomycin sulfate (4, 9). The Millipore filters and pads were, respectively, catalogue nos. AABPO4700 and AP100-4700. The cells were then kept in a moist atmosphere at 220. At intervals cells were harvested from the filter supports and assayed for cohesiveness; extracts were assayed for agglutination activity.D. discoideum strain A3 cells (mutant derived from NC-4) were grown in axenic culture (5). Growth-phase cells (vegetative) were tested for cohesiveness and extracts were assayed for agglutination activity.Cohesiveness Assay. Based on a procedure developed by Gerisch (6), we used roller-tube culturing to promote aggregation of cohesive slime-mold cells by passively bringing them into contact with one another independently of their chemotactic system. Particle-size analysis by a Coulter Electronic Particle Counter was used to determine the degree of aggregation. The procedure was as follows: (i) Harvested cells were washed twice in water at 40 and suspended in EDTA-phosphate buffer [16.7 mM Na2HPO4-KH2PO4, 10 mM EDTA (pH 6.2) ]. This buffer is slightly modified from one described by Gerisch (6). (ii) The suspension was dispersed into single cells by repeated pipetting through a fine-tipped pipette. Erythrocyte Agglutination Assay has been described (3). Briefly, the method was as follows: (i) Soluble crude extracts were prepared by homogenization of washed slime-mold cells 2554

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The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)-mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis.

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