Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors
Walczak et al. J Transl Med
Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors
Maciej P. Walczak 0 3
Anna M. Drozd 0 3
Ewelina Stoczynska‑Fidelus 0 2 3
Piotr Rieske 0 1 2 3
Dawid P. Grzela 0 3
0 Department of Research and Development, Celther Polska Ltd. , Milionowa 23, 93‐193 Łódź , Poland
1 Research and Development Unit, Personather Ltd. , Milionowa 23, 93‐193 Łódź , Poland
2 Department of Tumor Biology, Medical University of Łódź , Żeligowskiego 7/9, 90‐752 Łódź , Poland
3 Department of Research and Development, Celther Polska Ltd. , Milionowa 23, 93‐193 Łódź , Poland
Background: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. Methods: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline‑ inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. Results: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C‑ peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. Conclusions: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal‑ derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.
Defined culture conditions; Diabetes; Differentiation; Induced pluripotent stem cells; Insulin producing cells; NKX6; 1; PDX1; Reprogramming
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Type 1 diabetes (T1D) is one of the most frequent
chronic autoimmune diseases diagnosed among
juveniles, and its global incidence continues to rise [1]. This
condition is characterized by pancreatic beta cell
damage leading to insufficient insulin production and altered
carbohydrate metabolism. It develops at an early age and
requires constant treatment, which generates
substantial costs and lowers quality of life. Commonly, therapy
of type 1 diabetes is based on supplementation of
deficient hormone in form of regular injections. However,
this method does not address the cause of the disease,
regarded as a lack of functional intrinsic mechanisms
providing carbohydrate homeostasis.
Pancreatic islet transplantation is another approach to
T1D treatment. Its application is however limited due to
complex medical procedure and shortage in the number
of islets donors. Moreover, it is not a permanent
solution since patients who are forced to undergo several
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procedures eventually need to receive insulin injections
due to the destruction of the graft by the immune system
[2].
A concept of a therapy based on differentiated induced
pluripotent stem cells (iPSC) raises great promises in the
field of T1D treatment. These cells are generated from
somatic cells by forced expression of transcription factors
characteristic for embryonic stem cells [3]. Numerous
investigations have been conducted to confirm
resemblance between iPSC and embryonic stem cells (ESC)
isolated from the inner cell mass of developing embryo
[4, 5].
These cells are characterized by features of great
importance in terms of disease treatment, such as the
fact that they are obtained from patient’s own cells. This
trait eliminates risk of potential stem cell-based graft
rejection [6].
A number of research teams are focused on
generation of functional insulin producing cells (IPC) that could (...truncated)