Direct reprogramming of human fibroblasts into insulin-producing cells by transcription factors

Direct lineage reprogramming of one somatic cell into another bypassing an intermediate pluripotent state has emerged as an alternative to embryonic or induced pluripotent stem cell differentiation to generate clinically relevant cell types. One cell type of clinical interest is the pancreatic β cell that secretes insulin and whose loss and/or dysfunction leads to diabetes. Generation of functional β-like cells from developmentally related somatic cell types (pancreas, liver, gut) has been achieved via enforced expression of defined sets of transcription factors. However, clinical applicability of these findings is challenging because the starting cell types are not easily obtainable. Skin fibroblasts are accessible and easily manipulated cells that could be a better option, but available studies indicate that their competence to give rise to β cells through similar direct reprogramming approaches is limited. Here, using human skin fibroblasts and a protocol that ensures high and consistent expression of adenovirus-encoded reprogramming factors, we show that the transcription factor cocktail consisting of Pdx1, Ngn3, MafA, Pax4 and Nkx2-2 activates key β cell genes and down-regulates the fibroblast transcriptional program. The converted cells produce insulin and exhibit intracellular calcium responses to glucose and/or membrane depolarization. Furthermore, they secrete insulin in response to glucose in vitro and after transplantation in vivo. These findings demonstrate that transcription factor-mediated direct reprogramming of human fibroblasts is a feasible strategy to generate insulin-producing cells.


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The discovery that adult somatic cells can be reprogrammed into induced pluripotent 72 stem cells (iPSC) 1, 2 has revolutionized current biological and medical research.

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Expression relative to the housekeeping gene TBP was calculated using the delta(d)Ct 207 method and expressed as 2^(-dCT) unless otherwise indicated. Primer sequences are 208 provided in Table S3.

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Human insulin was measured using a human Insulin ELISA kit (Crystal Chem, 244 Zaandam, Netherlands) and C-peptide using a human C-peptide ELISA kit (Mercodia,

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We first investigated whether human fibroblasts could be directly converted towards the

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We chose adenoviruses for transgene delivery because they allow high expression of 333 transgenic proteins and, most importantly, don't integrate in the host genome.

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However, we couldn't detect Cherry immunofluorescence after viral treatment of human

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Together, these results reveal that 5TF induces a transcriptional switch from a 411 fibroblastic to a β-like cell identity that entails both selective gene activation and 412 repression events. Hereafter, we will refer to cells generated following the 5TF protocol 413 as induced β-like (iβ-like) cells.

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In vitro assessment of human fibroblast-derived iβ-like cells.

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4A and video S1). Parental HFF1 cells not engineered for 5TF expression were 424 universally unresponsive to these stimuli ( Fig. 4B and video S2). Among responsive 425 cells, approximately half responded to both glucose and high potassium and half responded only to potassium (Fig. 4A). We observed heterogeneity in the amplitude and kinetics of responses among individual cells (Fig. 4C). Next, we performed static 428 incubation assays to study glucose-induced insulin secretion (GSIS) and found that iβ-429 like cells released similar amounts of human insulin at low (2mM) and high (20mM) 430 glucose concentrations (Fig.4D). Thus, at this stage, iβ-like cells lack a key signature 431 feature of normal β-cells, the ability to increase insulin secretion in response to 432 changes in extracellular glucose.

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The differentiation and functionality of many cell types vary dramatically between three-

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In vivo assessment of human fibroblast-derived iβ-like cells.

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Lastly, we studied the maintenance of cellular reprogramming in vivo. To this end, we 475 transplanted 300 iβ-like cell spheroids (1000 cells/spheroid) into the anterior chamber 476 of the eye (ACE) of immune-deficient NOD scid gamma (NSG) mice (Fig. 6A,B). The

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ACE allows fast engraftment 44 and in vivo imaging 45 . To assess in vivo graft confirmed the presence of functional vessels in the grafts 10 days after transplantation ( Fig. 6C and video S1). We also established that the transplanted cells were viable by 481 visualizing fluorescence of the long-term tracer CFDA (Fig. 6C). We then investigated 482 whether reprogramming was retained in vivo. We first studied human INS gene 483 expression in eye grafts harvested ten days after transplantation. As shown in Figure   484 6D, human INS transcripts were readily detectable and levels (expressed relative to 485 TBP) were similar to those exhibited by iβ-like cells prior to transplantation ( Fig.2A, Fig.  levels observed in mice transplanted with 4000 iβ-like cell spheroids were in the same 508 order of magnitude than mice transplanted with a total of 300-500 human islets into the 509 ACE (Fig. 6H). Thus, correlating with determinations in the aqueous humor, these 510 results reveal that iβ-like cell clusters secrete in vivo approximately one tenth of the 511 amount of insulin released by human islets.

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In this study, we have developed a 10-day long protocol to produce insulin-producing . For example here we report that, by merely moving from 2D to a 3D culture 529 system, iβ-like cells acquired the ability to secrete insulin in response to glucose.

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Another simple strategy to improve the functionality of iβ-like cells would be the 531 addition of soluble factors during or after introduction of the conversion TFs 38, 53, 54 .
molecules that enhance maturation of β cells generated from stem cells 55 .

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Interestingly, it has been shown that soluble molecules can even substitute the function 535 of exogenous factors in some reprogramming protocols 56-58 .

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The   Supplementary S5: Immunostaining of iβ-cell spheroids ten days after their transplantation into the anterior chamber of the eye of NSG mice. Representative immunofluorescence images showing (A) C-peptide staining (green), HLA (red) and cell nuclei marked with Hoechst (white) and (B) C-peptide (green) with the indicated transcription factors (red). Scale bars are 25 µm.

Video S1
Video shows changes in fluorescence of an isolated iβ-like cell in response to high glucose (20mM) and high potassium (KCl 30mM). Cell had been pre-loaded with the calcium indicator Fluo-2. Video was recorded using a Leica TCS SPE confocal microscope with an incubation chamber set at 37 o C, and a 40X oil immersion objective.

Video S2
Video shows fluorescence of parental fibroblasts HFF1 in response to high glucose (20mM) and high potassium (KCl 30mM). Cells had been pre-loaded with the calcium indicator Fluo-2. Note that fibroblasts exhibit no changes in fluorescence in response to the tested stimuli. Video was recorded using a Leica TCS SPE confocal microscope with an incubation chamber set at 37 o C, and a 40X oil immersion objective.