Diabetes is a metabolic disease caused by the loss or impaired function of insulin-producing pancreatic β-cells. Different therapeutic strategies aim to restore the endogenous production of insulin rather than the cornerstone insulin injections treatment. Human pluripotent stem cells (hPSCs) have been proposed as an unlimited source for cell-based therapy of diabetes through the directed differentiation into functional pancreatic β cells. Step-wise differentiation protocols based on developmental biology of pancreas, have led to the generation of insulin-producing β cells. However, the majority of the cells produced were poly-hormonal as they expressed other hormones in addition to insulin and have failed to respond when challenged with glucose. The coordinate expression of particular transcription factors (TF) in distinct stages governs the differentiation of hPSCs into insulin β cells. Pancreatic and duodenal homeobox protein (PDX1) is a crucial TF required for pancreas development. On the other hand, homeobox protein NKX6.1 is a potent bi-functional TF that is essential for β cells maturation, proliferation and insulin metabolism. The dual expression of PDX1 and NKX6.1 during multipotent progenitor cell (MPC) stage is vital for guiding the cells towards functional β cells lineage. However, cells expressing PDX1 but lack NKX6.1 expression tend to take the poly-hormonal path. This guided the differentiation protocols to focus on enriching MPC population co-expressing PDX1 and NKX6.1. The aim of this study was to further explore different MPC populations in terms of PDX1/NKX6.1 expression. We used two different differentiation protocols to differentiate hESCs and hiPSCs into MPCs. The mRNA and protein expressions of the generated MPCs were analyzed using immunocytochemistry, RT-PCR, and flow cytometry. Our results showed that hPSCs were successfully differentiated into the conventional (PDX1+/NKX6.1+) and (PDX1+/NKX6.1-) MPC populations. The efficiency of differentiating hPSCs into PDX1+/NKX6.1+ MPCs has varied between the two used protocols. Immunofluorescence staining has unveiled the generation of a novel population that expressed NKX6.1 independently of PDX1 (PDX1-/NKX6.1+) in both hESCs and hiPSCs. This is surprising considering that PDX1 was reported to bind to the promoter of NKX6.1 gene and is needed for NKX6.1 expression. Furthermore, using our optimized protocol, this uncharacterized subset of MPCs was enriched and found to exhibit a pattern of three-dimensional (3D) aggregates that were consistently (PDX1-/NKX6.1+) and surrounded by either (PDX1+/NKX6.1+) or (PDX1+/NKX6.1-) MPCs. To understand and characterize this unique population, we examined the expression of other TFs including endocrine precursors markers Chromogranin A (CHGA) and NKX2.2. CHGA was found to be expressed in the same areas that were positive for NKX6.1 and PDX1. However, the 3D structures that were PDX1-/NKX6.1+ did not co-express CHGA. On the contrary, few cells of these 3D aggregates co-expressed NKX2.2, suggesting that this population may have an undefined role in the development of MPCs into endocrine progenitors. These findings showcase a novel population of NKX6.1 expressing MPCs that did not require PDX1 expression at this stage. Moreover, this population may retain an alternative path towards pancreatic islet cells development that is independent of PDX1. A thorough characterization of this population is needed to explore the regulatory gene network controlling their lineage specification.


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