## 4 Discussion  
Transplantation of neonatal porcine islets-like clusters (NPICCs) has the potential to overcome the problem of a shortage of organ donors. Optimization of pretransplant islet culture conditions is of great importance to increase the number of high quality, functionally active islets. In the present study, we investigated the potential of SCFAs to stimulate NPICCs development and the maturation of pancreatic beta cells. We demonstrated that butyrate and class I specific HDACi strongly induced insulin expression in NPICCs, which provides novel options to improve cell replacement therapies. Because several studies have shown that SCFAs may directly promote survival and function of human and rodent pancreatic beta cells [16; 22; 23; 24], we tested whether acetate, butyrate and propionate can accelerate differentiation and maturation of cells residing in immature NPICCs from 2-3 days old piglets. Consistent with previous reports, we observed a spontaneous increase of the percentage of alpha and beta cells from day three to day nine cultures [8; 11; 25; 26]. We here report that butyrate but not acetate or propionate has the potential to activate a maturation program and significantly increase gene and protein expression of beta cell maturation markers compared to our standard culture protocol. After butyrate treatment, NPICCs consist of about 65% hormone positive islet  
Figure 4: Inhibition of class I HDACs is involved in up-regulation of insulin gene expression. (**A**) Butyrate dose-dependently reduces in vitro HDAC enzyme activity in a HDAC fluorometric assay. (**B**) NPICCs were treated with 1000 \(\upmu\)M butyrate and HDAC activity was measured by HDAC Activity Fluorometric Assay. Butyrate (1000 \(\upmu\)M) and trichostatin A (TSA, 1 \(\upmu\)M) significantly inhibited HDAC enzyme activity. (**C**) NPICCs were treated with different HDAC inhibitors for 6 days. TSA (1 \(\upmu\)M) (class I and class II inhibitor), mocetinostat (1 \(\upmu\)M) and MS275 (1 \(\upmu\)M) (selective class I inhibitors) but not TMP269 (1 \(\upmu\)M) or MC1568 (1 \(\upmu\)M) (selective class II inhibitors) significantly increased insulin gene expression. NPICCs cultured in B-IC medium were used as controls. Data from three to five independent experiments are presented as mean \(\pm\) SD. \({}^{*}p<0.05\), \({}^{**}p<0.01\), \({}^{***}p<0.001\), \({}^{***}p<0.0001\) vs. control groups. cells including 43% insulin-, 12% glucagon-, and 10% somatostatin-positive cells. The observed increase in the number of beta cells could be explained by replication of pre-existing beta cells and/or the differentiation of residing islet precursor cells [10; 26]. Since we did not detect an increased proliferation rate, it is likely that butyrate mainly induced development of new beta cells from ductal or progenitor-like cells. Lineage tracing studies of ductal and progenitor cells are necessary to clarify this question. Earlier reports described a two to threefold increase of insulin expression with 23% insulin positive cells in porcine fetal cell clusters cultured for one week in medium supplemented with 10% human serum with either nicotinamide or butyrate [25]. [PERSON] described a three to fivefold increased insulin content and threefold increased insulin gene expression in hydrogel encapsulated NPICCs after 14 days incubation in medium with 10% FCS stimulated with 500 uM butyrate and 10 nM IGF-1 plus/minus nicotinamide [14]. In our NPICC isolation protocol we exclude serum supplement, because serum growth factors are not well defined, and it leads to an attachment of NPICCs on the plastic surface of the culture dishes followed by a spreading of attached cells and rapid loss of expression of endocrine and beta cell markers (data not shown). A similar high number of endocrine cells was reported in a recent study, which shows that a 20-days culture period of NPICCs in medium with sequential addition of dexamethasone and oncostatin M, nicotinamide/exendin-4, and TGF-beta1/thrombin generated 42% insulin and 17% glucagon positive cells [11].