Reprogramming with hyperglycaemia leads to fewer insulin+ cells
To determine if hyperglycaemia has a negative influence on reprogramming, STZ-treated diabetic Rag
−/− mice were randomly assigned to a normoglycaemic group by transplantation of 500 islets of C57-mice or a hyperglycaemic group by implantation of InsP to keep animals viable yet hyperglycaemic. M3Cherry or Cherry control viral constructs were injected into the pancreas 2 weeks later, and animals were killed after 10 or 25 days (Fig. 1a). Mice treated with InsP stayed hyperglycaemic (30.5 ± 0.4 mmol/l, n = 18), while transplanted animals were normoglycaemic (9.2 ± 0.3 mmol/l, n = 27; Fig. 1b). Regardless of glucose levels, reprogramming with the M3Cherry viral construct led to the appearance of single or clustered insulin+ cells in the pancreatic tail with minimal numbers of Cherry+ cells stained for glucagon, somatostatin or pancreatic polypeptide (Fig. 1c, ESM Fig. 1a–c). On day 10, recipients of islet transplants had 141 ± 22 × 103 insulin+ cells, while InsP recipients had 84 ± 20 × 103 (both n = 5; p = 0.2, Fig. 1d, ESM Fig. 2a). The number of reprogrammed insulin+ cells in normoglycaemic animals increased from day 10 to 25 (233 ± 25 × 103, n = 4) in contrast to the hyperglycaemic group with an unchanged number of reprogrammed cells (79 ± 10 × 103, n = 5, Fig. 1d). Thus, the estimated total number of reprogrammed insulin+ cells differed significantly in hyperglycaemic and normoglycaemic animals on day 25 (p < 0.05, Fig. 1d). For individual animals at each time point (day 10 and 25), there was an inverse correlation between mean glucose levels after reprogramming and the number of reprogrammed insulin+ cells (day 10: r = −0.75, p = 0.003 [n = 13]; day 25: r = −0.94, p = 0.02 [n = 6], ESM Fig. 2b,c). This inverse correlation even existed in normoglycaemic mice receiving mouse or rat islets, the latter having lower glucose levels due to rat beta cells having lower glucose set-points for secretion (insert ESM Fig. 2b).
With hyperglycaemia, reprogrammed cells show preserved reprogramming initiation but then less differentiation toward a beta cell phenotype
We assessed the temporal sequence of reprogramming events. Cell size reduction has been described as an early morphological response to iPS cell and exocrine to beta cell reprogramming [17, 33]. Moreover, we have previously shown that acinar to beta cell reprogramming follows a temporal sequence with acinar cells adopting first an endocrine and then beta cell fate . Thus, reprogramming events were followed over time: (1) initiation of reprogramming as assessed by cell size reduction; (2) endocrine commitment by chromogranin A (ChgA) expression; and (3) beta cell commitment by insulin expression.
Maximal diameters of Cherry+ cells were determined in M3Cherry-injected animals and Cherry-injected controls. In both hyperglycaemic (hyper) and normoglycaemic (normo) conditions, cell sizes of Cherry+insulin+ (hyper 18.1 ± 0.5 μm, normo 18.3 ± 0.4 μm) as well as Cherry+insulin− (hyper 17.5 ± 0.6 μm, normo 18.3 ± 0.4 μm) cells were significantly smaller in M3Cherry-treated animals compared with Cherry+ cells in Cherry-injected controls (hyper 27.7 ± 0.4 μm, normo 28.7 ± 0.2 μm, all comparisons p < 0.05, Fig. 2a), indicating preserved reprogramming initiation by cell size reduction, irrespective of glucose levels.
The endocrine commitment as assessed by ChgA expression was 91.2 ± 2.3% of Cherry+ cells in hyperglycaemic mice on day 10, while virtually all Cherry+ cells co-expressed ChgA in normoglycaemic animals (99.0 ± 0.4%, p = 0.008; Fig. 2b), suggesting impaired endocrine commitment with hyperglycaemia. This difference in ChgA expression was also seen on a qualitative level, as ChgA expression in hyperglycaemic animals was much weaker and more inhomogeneous compared with its strong and even expression in normoglycaemia.
Beta cell commitment was assessed by the proportion of infected Cherry+ cells stained for insulin. On day 10, the percentage of insulin+/Cherry+ cells was significantly lower in hyperglycaemic (37.2 ± 4.9%, n = 5) compared with normoglycaemic mice (57.3 ± 5.8%, n = 5; Fig. 2c). While by day 25 in hyperglycaemia the percentage of insulin+/Cherry+ cells only increased to 57.0 ± 4.5% (n = 5), with normoglycaemia reprogrammed cells exhibited a reprogramming efficiency of 79.0 ± 6.0% insulin+/Cherry+ cells (n = 3), indicating increasing commitment toward the beta cell lineage (Fig. 2c). This went along with significantly lower expression of NKX2.2 (NK2 homeobox 2) protein in reprogrammed cells of hyperglycaemic animals (72.2 ± 5.8%, n = 3) compared with normoglycaemic animals (87.3 ± 3.3%, n = 3, p < 0.05, t test). Taking these findings together, hyperglycaemia does not affect reprogramming initiation as reprogrammed cells still undergo cell size reduction, yet inhibits the endocrine and beta cell commitment later in this temporal sequence.
Normoglycaemia during reprogramming is required for function of reprogrammed cells
When M3Cherry viral constructs were injected into hyperglycaemic animals that had received InsP-implants, sustained normalisation of glucose levels was not achieved (not shown). To determine at what stage hyperglycaemia affects the function of reprogrammed cells, nephrectomies (Nx) of the kidneys carrying the islet grafts were performed early (day 10) or late (day 25) after reprogramming. After Nx, glycaemia fully depended on reprogrammed cells as endogenous beta cells had previously been ablated by STZ (Fig. 3a). Hyperglycaemia defined as glucose >11.1 mmol/l in the 2 weeks following Nx occurred in 5/6 animals in the early Nx group while 1/6 animal was hypoglycaemic as opposed to 2/7 hyperglycaemic, 3/7 normoglycaemic and 2/7 hypoglycaemic animals in the late Nx group. Glucose levels at 1 week after Nx were 21.7 ± 3.9 mmol/l in the early Nx group (n = 6), which was significantly higher than the levels of 7.9 ± 1.8 mmol/l (n = 7) in the late Nx group (Fig. 3b, c). Thus, hyperglycaemia during reprogramming results also in functional impairment of insulin+ cells, while their function is maintained in a normoglycaemic environment.
Reprogramming in hyperglycaemia leads to changes in the exocrine cell compartment with prolonged inflammatory tissue remodelling
With hyperglycaemia and the M3Cherry injection, major tissue remodelling in the exocrine cell compartment was evident on day 10, with formation of abundant tubular complexes reminiscent of those seen in pancreatitis . Insulin+ cells were found contained within these tubular structures (Fig. 4a, b, ESM Fig. 3). Cells of tubular complexes strongly expressed the mitochondrial stress marker HSP60 in hyperglycaemic animals (ratio of HSP60+:Cherry+ cells 3.6 ± 0.5, n = 5). Such tubular complexes were not found in normoglycaemic M3Cherry-injected animals at this time point (ratio of HSP60+:Cherry+ cells 1.2 ± 0.1, n = 5; Fig. 4a, ESM Fig. 3), and by day 25 they were no longer predominant in either group (ratio HSP60+:Cherry+ cells 1.0 ± 0.01 [hyper, n = 3] and 1.1 ± 0.05 [normo, n = 3]; Fig. 4a).
panCK(pan-cytokeratin), a marker of pancreatic duct epithelial cells, was found to be strongly expressed in the tubular complexes of hyperglycaemic M3Cherry-injected animals on day 10 (ratio panCK+:Cherry+ cells 3.9 ± 0.6 [hyper, n = 5] vs 0.6 ± 0.08 [normo, n = 5]), while this differential was gone by day 25 (ratio panCK+:Cherry+ cells 1.0 ± 0.2 [hyper, n = 4] vs 0.4 ± 0.02 [normo, n = 4]; Fig. 4b, ESM Fig. 3). As with HSP60 expression, panCK+ tubular complexes were not found in hyperglycaemic Cherry-injected controls and noninfected areas of M3Cherry-injected animals (not shown). With normoglycaemia, the majority of reprogrammed cells were found in clusters potentially representing former exocrine acini. This arrangement was less pronounced with hyperglycaemia, consistent with disrupted pancreatic architecture (Fig. 2c). Together, these findings support the notion that hyperglycaemia prolongs the time of inflammatory exocrine tissue remodelling.
Rapid downregulation of the exocrine gene programme is known to precede adoption of an insulin+ phenotype . Thus, impaired reprogramming in hyperglycaemia might be due to a failure of acinar cells to efficiently downregulate their exocrine cell programme. Indeed, we found higher expression of PTF1A (pancreas specific transcription factor, 1a) protein (Fig. 4c) in reprogrammed cells of hyperglycaemic (20.4 ± 5.3%, n = 3 insert) relative to that in normoglycaemic animals by day 10 (9.3 ± 2.8%, n = 3, p < 0.05, t test). These findings provide additional information about the adverse effects of hyperglycaemia on reprogramming.
Exocrine tissue remodelling is associated with a transient macrophage influx in infected areas
Microarray analyses were performed on Cherry+ cells harvested 7 days after reprogramming. Strikingly, differences in gene expression predominantly concerned inflammatory markers, which were more prominent in the hyperglycaemic condition. By gene set enrichment analysis, we found 25 enriched gene sets in hyperglycaemia defined by an FDR <25% to account for multiple testing, 13 of which were directly connected with inflammation or apoptosis (Table 1).
Therefore, we examined the involvement of immune cells in the reprogramming failure of hyperglycaemic animals. As Rag
−/− mice do not harbour mature B and T lymphocytes, we focused on the role of macrophages. Immunohistochemistry revealed increased numbers of macrophages as assessed by F4/80 staining in areas of reprogramming in hyperglycaemic compared with normoglycaemic M3Cherry-injected animals on day 10 (F4/80+ cells/mm2: 1,501 ± 263 [hyper, n = 5] vs 625 ± 65 [normo, n = 4]; Fig. 5a). On day 25, however, the number of macrophages had declined in both glycaemic conditions (F4/80+ cells/mm2: 562 ± 158 [hyper, n = 4] vs 268 ± 23 [normo, n = 4]; Fig. 5a), demonstrating the transient nature of the inflammatory response. Macrophages were sparse in hyperglycaemic Cherry-injected controls and in uninfected areas of M3Cherry-injected animals (uninfected areas F4/80+ cells/mm2: 69 ± 15 [hyper, n = 5] vs 77 ± 15 [normo, n = 4] day 10; 30 ± 8 [hyper, n = 5] vs 30 ± 5 [normo, n = 4] day 25). Double-staining for tubular complexes and macrophages revealed their spatial relationship with macrophages adjacent to the tubular structures (ESM Fig. 4).
Macrophages in the infected pancreas clear tissue from partially reprogrammed cells
If macrophages were directly deleterious to reprogramming success, reducing their number during or after viral delivery of transcription factors could give rise to more reprogrammed cells. We established a model of macrophage depletion by whole body irradiation, followed by bone marrow Tx of CD11b-DTR or wild-type (WT) donors and DT injections for 10 days to ablate CD11b-DTR cells (Fig. 5b). The 10 day time point was analysed for macrophage infiltration and reprogramming efficiency. As expected, WT transplanted mice had significantly higher macrophage infiltration (macrophages/mm2 640 ± 120 [SD], n = 4) compared with CD11b-DTR transplanted mice treated with DT (macrophages/mm2 333 ± 71 [SD], n = 4; Fig. 5c). mRNA isolated from infected pancreas confirmed reduced presence of macrophages by lower Emr1 (also known as Adgre1) expression (the gene product encoding F4/80 antigen) in CD11b-DTR transplanted mice, whereas adenoviral M3Cherry was expressed at similar levels (Fig. 5c). We also found strong mRNA downregulation (>20-fold) of the M2-macrophage polarisation marker Retnla (encoding resistin like alpha) in CD11b-DTR transplanted mice, concomitant with a sevenfold upregulation of Tnfα in the same tissue (ESM Fig 5). This finding indicates that primarily M2-polarised macrophages were depleted, and that their eradication exacerbated the inflammatory milieu. Confirming efficient monocyte/ macrophage depletion, the relative percentage of CD11b+/Gr1−/low monocyte relative to CD11b+/Gr1high granulocytes was significantly lower in the peripheral blood in macrophage-ablated animals (ESM Fig. 5). However, macrophage-depleted animals did not display increased reprogramming efficiency compared with controls (insulin+/Cherry+ cells, CD11b-DTR: 69 ± 5% [SD], WT: 84 ± 5% [SD]; Fig. 5c). We conclude that at least under normoglycaemia, macrophages are not directly detrimental to reprogramming and might even engage in dampening tissue inflammation.
To better understand the role of macrophages, we assessed their spatial relationship with reprogrammed cells and found that on day 25 a substantial number of macrophages had engulfed Cherry+insulin− cells, which was most pronounced in the hyperglycaemic group (macrophages with Cherry+insulin− 36.2 ± 5.1% [hyper, n = 4] vs 19.7 ± 4.8% [normo, n = 4]; insert Fig. 5d). The numbers of engulfed Cherry+insulin+ cells in macrophages were generally lower (Fig. 5d). TUNEL staining to assess whether reprogrammed cells had undergone apoptosis prior to engulfment by macrophages showed that occurrence of apoptotic Cherry+ cells was rare in both hyperglycaemic and normoglycaemic conditions, yet slightly higher in hyperglycaemic animals on day 10 (p = 0.03; not shown). Thus, low apoptosis rate and at the same time high numbers of engulfed Cherry+ cells in macrophages suggests that either apoptosis is not a prerequisite for macrophage engulfment or that apoptosis is difficult to capture. Thus, in the presence of hyperglycaemia it appears that a high number of macrophages are recruited by day 10 due to the presence of incompletely reprogrammed cells, some of which are found to be engulfed by the macrophages at day 25.