Hybrid cells survive and grow in double hygromycin/neomycin selection medium
To generate somatic cell hybrids, G418-resistant βlox5 were fused with hygromycin-resistant MIN6 using polyethylene glycol (Supplementary Figure 1
). Cells were then cultured continuously in G418/hygromycin selection medium. A fusion between G418-resistant and hygromycin resistant MIN6, done as a control for effects of fusion on differentiation, was efficient, with hundreds of colonies per plate. Surviving cells were pooled in an uncloned population designated MMC. Fusion of MIN6 cells with βlox5 was much less efficient, (one event per 1x106
human cells). Only five stable clones were generated, each from a different well. Each clone was cultured continuously through approximately 40 doublings. Three representative hybrid clones, BMC1-3, were further analysed.
Inhibition of mouse insulin gene transcription
Initial PCR analysis examined mouse and human insulin mRNA using species-specific primers. Since chromosome loss occurs in mouse-human cell fusions [4
], leading to inter- and intra-clonal heterogeneity, we also used PCR to detect genomic DNA in hybrid and parental cells, ensuring the presence of all genes of interest (Supplemental Fig. 2
). Thus, we confirmed that interspecies fusion had occurred, and that each hybrid clone contained the gene of interest, so that RNA gene transcript changes reflected a transcriptional event rather than chromosome loss.
As expected, parental MIN6 homologous fusions (MMC) retained high levels of mouse insulin I and II gene transcripts (), indicating that cell fusion by itself was not detrimental. In contrast, neither βlox5 nor any mixed hybrid BMC clones expressed human insulin (). Mouse insulin I and insulin II expression was abolished in the BMC clones, except for a low level of insI mRNA in BMC1. Thus, a gene or genes expressed in βlox5 appear to dominantly repress insulin gene expression.
Figure 1 A. RT-PCR analysis of human and mouse insulin and insulin transcription factor mRNA. GAPDH was used to ensure that equal amounts of cDNA were used. B. Immunostaining for insulin (red in left panels) and PDX1 (red in right panels). Nuclei are labeled by (more ...)
Reprogramming of transcription factors in insulin-producing cells following cell fusion
Expression of PDX1, NeuroD and MafA was analyzed as they are important transactivators of insulin gene expression [5
]. MIN6 and homologous Min6 MMC fused cells expressed high levels of mRNA for each of these factors (), but neither βlox5 nor hybrids expressed mRNA for human PDX1, MafA, or NeuroD1 ().
Each hybrid clone expressed detectable levels of the mouse PDX1 gene transcript, but the levels of expression were lower than in MIN6 or MMC cells (). Mouse MafA was weakly detectable in each hybrid clone, while mouse NeuroD was detectable only in the hybrid BMC1 (). Notably, the only hybrid clone expressing detectable mouse PDX1, NeuroD and MafA mRNA was BMC1, which was also the only clone retaining expression of mouse insulin I gene transcripts. Consistent with the gene expression data, BMC1 expressed PDX-1, NeuroD1, and MafA protein, but only at a low level, resembling the pattern in βlox5 much more closely than that in MIN6 ().
Hybrid clones lose insulin and PDX1 immunostaining
To determine whether the low level of mouse INS1 and PDX-1 mRNA in BMC1 was due to homogeneous extinction by a dominant factor in βlox5, or whether there was heterogeneity in the BMC1 cells despite their having arisen from a single colony, immunostaining for those proteins was used. MIN6 and MMC cells all strongly expressed insulin, while neither βlox5 nor BMC1 displayed any insulin immunostaining, as expected ().
Like insulin, PDX1 protein was strongly expressed in MIN6 and MMC cells and absent in βlox5 (). However, the BMC1 hybrid had a small population of strongly PDX1 positive cells ().
Notch activation is dominant in human-mouse somatic cell hybrids
Notch signalling plays an important role in pancreatic differentiation and cell fate decisions [6
]. Overexpression of Notch1 leads to dedifferentiation of INS1 insulinoma cells as assessed by reduction of both PDX1 and insulin gene transcription [9
]. Hes1 also mediates repression of insulin gene transcription in dexamethasone treated HIT insulinoma cells [10
]. Thus it was a logical candidate for a regulatory role in the reprogramming of the insulin gene program in hybrid cells. Hes1 is the key downstream effector of the Notch signalling pathway in β-cells [11
]. To determine if Hes1 was significant in regulating the differentiation state of pancreatic cell lines, we performed quantitative real-time RT-PCR on RNA from the parental cell lines MIN6 and βlox5, revealing that the non-insulin-producing βlox5 cells had Hes1 transcript levels 10-fold higher than insulin-producing MIN6 cells relative to GAPDH, using a set of primers that detected both human and mouse GAPDH (). Because Hes1 is controlled at both transcriptional and post-transcriptional levels, Western blots for Hes1 were performed, showing that the βlox5 and hybrid cells had considerably higher levels of Hes1 protein than MIN6 cells ().
A. Quantitative real-time RT-PCR of Hes1 and Hes6 mRNA in parental cell lines.
Based on the pattern of Hes1 expression in the parental cell lines, Notch signalling became a candidate for a dominant factor that repressed β-cell differentiation in the hybrids. If so, Hes1 levels in the BMC hybrids should be similar to those in βlox5 rather than Min6. RT-PCR analysis () and Western blot analysis (), demonstrated that the state of Notch activation in βlox5 is dominant over that in Min6.
The Notch pathway inhibitor Hes6 is highly expressed in islets and is differentially expressed in βlox5, Min6, and hybrids
To determine whether Hes1 was playing an active role in the nuclear reprogramming and repression of differentiation in the hybrid clones, it was necessary to repress Hes1 function. In a number of cell systems, Hes6 inhibits Hes1. The genes share substantial sequence similarity to Hes1 except in the loop region within the bHLH domain. [12
]. Unlike Hes1, Hes6 does not bind to N-box sequences and in fact has been found to act as a potent repressor of Hes1 by multiple mechanisms, including inhibiting Hes1 binding to coactivators and by targeting Hes1 protein for degradation [13
The role of Hes6 in the pancreas has not been studied in depth. Immunohistochemistry revealed that Hes6 is highly expressed in islets. Strikingly, it is limited to β-cells, being absent in α-, δ-, and PP cells (). Consistent with a role for Hes6 in maintaining β-cell differentiation, both Western blot () and quantitative RT-PCR () revealed that insulin-producing MIN6 cells expressed a higher level of Hes6 than did βlox5 cells.
Both mouse and human Hes6 mRNA levels dropped substantially in the hybrids compared with the parental cells () and was consistent with Western blot analysis, which found that βlox5 and BMC1 cells had markedly lower levels of Hes6 than MIN6 ().
Transduction with Hes6 retrovirus restores insulin expression in human/mouse hybrids
The Hes6 and Hes1 expression patterns were consistent with, but did not prove, that Hes1 and Hes6 were causal in modulating β-cell differentiation. If they were, we hypothesized that elevating the level of Hes6 would reverse the dedifferentiation observed in the human/mouse hybrids. To express Hes6, its cDNA was inserted into a retroviral vector. In βlox5, quantitative RT-PCR analysis demonstrated that the level of Hes6 mRNA increased substantially (), and immunostaining with an anti-Hes6 antibody revealed that the level of Hes6 protein in infected cells was markedly increased compared with uninfected cells (), but there was no significant effect on β-cell differentiation. In Min6, Hes6 overexpression decreased the level of Hes1 mRNA by 54% (P<0.05; ) and reduced the level of Hes1 protein as well (). PDX1, NeuroD and MafA mRNA levels were all significantly increased (2.7-, 1.7-, and 1.6-fold respectively, P<0.05), but the levels of mouse insulin I and II mRNA were unchanged, suggesting that neither the insulin transactivators nor Hes1 are limiting insulin gene expression (). However, exogenous Hes6 expression had a striking effect on BMC1 cells, where there was a substantial increase in the level of mouse insulin I mRNA (6.9-fold, P<0.05; ). In those cells Hes6 reduced the levels of both mouse and human Hes1 mRNA (39% and 42% decrease respectively, P<0.05; ). Mouse PDX1, NeuroD, and MafA mRNAs were significantly increased (1.8-, 1.5-, and 2.3-fold respectively, P<0.05; ), but human PDX1, NeuroD, MafA, and insulin mRNA levels were either absent or unchanged. Overexpression of Hes6 in the BMC2 and BMC3 hybrids, which had completely lost endogenous PDX-1 expression, had no effect on the pattern of either murine or human gene expression.
Figure 3 Effects of infecting parental βlox5 cells with retrovirus expressing Hes6 were determined by: A. Quantitative RT-PCR showing levels of Hes6 normalised to GAPDH (representative of three experiments), B. Immunostaining for Hes6, C. Western blotting (more ...)
Figure 4 Quantitative RT-PCR showing the effects of Hes6 on insulin expression. Hes1 (A), PDX1, NeuroD and MafA (B) in uninfected (−) and infected (+) MIN6 and BMC1 cells. Data are expressed as relative mRNA levels normalised to GAPDH and represent mean (more ...)
As described above, PDX-1 continued to be expressed at a high levels in a subset of BMC1 hybrid cells (, ). Thus, it was of interest to determine whether the increased insulin in the hybrid cells infected with the Hes6 vector was restricted to those expressing a high level of PDX-1. BMC1 hybrids infected with the Hes6 virus exhibited an increased number of cells expressing PDX1 protein compared with uninfected cells (), and most notably, a significant number of the PDX1-positive cells in the Hes6-infected hybrid cells demonstrated colocalization with insulin (). Thus, a subset of cells in BMC1, i.e., those expressing PDX-1, were highly responsive to Hes6, exhibiting a large increase in insulin expression. The cells not expressing PDX-1 did not stain positively for insulin. This means that the RT-PCR data on the effects of Hes6 most likely underestimate the effects as they include both cells expressing and not expressing PDX-1. Overall, these data indicate that it is possible to partially restore the differentiation state of these cells by increasing Hes6 expression.