In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to up-regulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor.

https://www.pnas.org/content/pnas/109/19/7517.full.pdf

Krüppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract

The number of patients worldwide suffering from the chronic disease diabetes mellitus is growing at an alarming rate. Insulin-secreting β-cells in the islet of Langerhans are damaged to different extents in diabetic patients, either through an autoimmune reaction present in type 1 diabetic patients or through inherent changes within β-cells that affect their function in patients suffering from type 2 diabetes. Cell replacement strategies via islet transplantation offer potential therapeutic options for diabetic patients. However, the discrepancy between the limited number of donor islets and the high number of patients who could benefit from such a treatment reflects the dire need for renewable sources of high-quality β-cells. Human embryonic stem cells (hESCs) are capable of self-renewal and can differentiate into components of all three germ layers, including all pancreatic lineages. The ability to differentiate hESCs into β-cells highlights a promising strategy to meet the shortage of β-cells. Here, we review the different approaches that have been used to direct differentiation of hESCs into pancreatic and β-cells. We will focus on recent progress in the understanding of signaling pathways and transcription factors during embryonic pancreas development and how this knowledge has helped to improve the methodology for high-efficiency β-cell differentiation in vitro.

Stem Cells to Pancreatic β-Cells: New Sources for Diabetes Cell Therapy

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

Due to the limited supply of donor pancreas, it is imperative that we identify alternative cell sources that can be used to treat diabetes mellitus (DM). Multipotent adipose tissue–derived stem cells (ADSC) can be abundantly and safely isolated for autologous transplantation and therefore are an ideal candidate. Here, we report the derivation of insulin-producing cells from human or rat ADSC by transduction with the pancreatic duodenal homeobox 1 (Pdx1) gene. RT-PCR analyses showed that native ADSC expressed insulin, glucagon, and NeuroD genes that were up-regulated following Pdx1 transduction. ELISA analyses showed that the transduced cells secreted increasing amount of insulin in response to increasing concentration of glucose. Transplantation of these cells under the renal capsule of streptozotocin-induced diabetic rats resulted in lowered blood glucose, higher glucose tolerance, smoother fur, and less cataract. Histological examination showed that the transplanted cells formed tissue-like structures and expressed insulin. Thus, ADSC-expressing Pdx1 appear to be suitable for treatment of DM.

Treatment of Type 1 Diabetes with Adipose Tissue-Derived Stem Cells Expressing Pancreatic Duodenal Homeobox 1

https://pathology.ufl.edu/files/2012/08/tang-dq-2006-lab-inv.pdf

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors.

Objective: The key pancreatic transcription factor Pdx1, known to control development and maintenance of pancreatic β-cells, possesses a protein transduction domain (PTD) that facilitates its entry into cells. We therefore sought to evaluate the capacity of in vivo -administered recombinant Pdx1 (rPdx1) to ameliorate hyperglycemia in a streptozotocin (Stz)-induced diabetic mice.

Research Design and Methods: Cell entry and transcriptional regulatory properties of rPdx1 and its PTD-deletion mutant rPdx1Δ, as well as a PTD-GFP reporter protein were evaluated in vitro . After intraperitoneal rPdx1 injection into Stz-diabetic mice, we assessed the its action on blood glucose levels, insulin content, and IPGTT, as well as Pdx1 distribution, pancreatic gene expression, islet cell proliferation, and organ histology.

Results: Restoration of euglycemia in Pdx1-treated diabetic mice was evident by improved IPGTT and glucose-stimulated insulin release. Insulin-, glucagon-, and Ki67-immunostaining revealed increased islet cell number and proliferation in pancreata of rPdx1-treated mice. Real-time PCR of pancreas and liver demonstrated up-regulation of INS and PDX1 genes as well as other genes relevant to pancreas regeneration. While the time-course of β-cell gene expression and serum/tissue insulin levels indicated that liver- and pancreas-derived insulin contributed to restoration of normoglycemia, near-total pancreatectomy resulted in hyperglycemia, suggesting that β-cell regeneration played the primary role in rPdx1-induced glucose homeostasis.

Conclusions: rPdx1 treatment of Stz/diabetic mice promotes β-cell regeneration and liver-cell reprogramming, leading to restoration of normoglycemia. This novel PTD-based protein therapy offers a promising way to treat patients with diabetes, while avoiding potential side-effects associated with the use of viral vectors.

/pdf/reversal-of-streptozotocin-induced-diabetes-in-mice-by-5b0qqnmhxz.pdf

Reversal of streptozotocin-induced diabetes in mice by cellular transduction with recombinant pancreatic transcription factor pancreatic duodenal homeobox-1: a novel protein transduction domain-based therapy.

Ectopic expression of Pdx1 triggers rapid hepatocyte dedifferentiation by down-regulating liver-enriched transcription factors and liver-specific functional genes such as hepatic nuclear factor-1α (HNF1α), albumin, and AAT. However, the links between Pdx1 over-expression and hepatic gene down-regulation are incompletely understood. HNF1α and HNF4α are important transcription factors that establish and maintain the hepatocyte phenotype. The human HNF4α gene contains two promoters (P1 and P2) that drive expression of P1-(HNF4α 1-6) or P2-(HNF4α 7-9)-derived isoforms, which are used in different tissues and at different times during development. We hypothesized that the relative expression of HNF1α and HNF4α following ectopic Pdx1 expression may promote hepatic cell dedifferentiation and transdifferentiation toward pancreatic beta-cells. We produced lentiviruses expressing Pdx1, Pdx1-VP16, and Ngn3, along with dual-color reporter genes to indicate hepatic and pancreatic beta-cell phenotype changes. Using these PTF alone or in combinations, we demonstrated that Pdx1 not only activates specific beta-cell genes but down-regulates HNF1α. Pdx1-mediated reduction of HNF1α is accompanied by altered expression of its major activator, HNF4α isoforms, down-regulating hepatic genes ALB and AAT. Pdx1 up-regulates HNF4α via the P2 promoter. These P2-driven isoforms compete with P1-driven isoforms to suppress target gene transcription. In Huh7 cells, the AF-1 activation domain is more important for transactivation, whereas in INS1 cells, the F inhibitory domain is more important. The loss and gain of functional activity strongly suggests that Pdx1 plays a central role in reprogramming hepatocytes into beta-cells by suppressing the hepatic phenotype.

Pancreatic and duodenal homeobox gene 1 (Pdx1) down-regulates hepatic transcription factor 1 alpha (HNF1α) expression during reprogramming of human hepatic cells into insulin-producing cells.

Shun Lu

Papers

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors.

Reversal of streptozotocin-induced diabetes in mice by cellular transduction with recombinant pancreatic transcription factor pancreatic duodenal homeobox-1: a novel protein transduction domain-based therapy.

Pancreatic and duodenal homeobox gene 1 (Pdx1) down-regulates hepatic transcription factor 1 alpha (HNF1α) expression during reprogramming of human hepatic cells into insulin-producing cells.