Digestive Cells Converted into Insulin-Secreting Cells


By switching off a single gene, Columbia Medical Center scientists have converted cells from the digestive tract into insulin-secreting cells. This suggests that drug treatments might be able to convert gut cells into insulin-secreting cells.

Senior author Domenico Accili said this of this work: “People have been talking about turning one cell into another for a long time, but until now we hadn’t gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target.”

Accili’s work suggests that lost pancreatic beta cells might be replaced by retraining existing cells rather than transplanting new insulin-secreting cells. For nearly two decades, scientists have been trying to differentiate a wide variety of stem cells into pancreatic beta cells to treat type 1 diabetes. In type 1 diabetes, the patient’s insulin-producing beta cells are destroyed, usually by the patient’s own immune system. The patient becomes dependent on insulin shots in order to survive.

Without insulin, cells have no signal to take up sugar and metabolize it. Also muscles and the liver do not take up amino acids and make protein, and the body tends to waste away, ravaged by high blood sugar levels that progressively and relentlessly damage it without the means to repair this damage.

Insulin-producing beta cells can be made in the lab from several different types of stem cells, but the resulting beta cells often do not possess all the properties of naturally occurring beta cells.

This led Accili and others to attempt to transform existing cells into insulin-secreting beta cells. In previous work, Accili and others demonstrated that mouse intestinal cells could be converted into insulin-secreting cells (see Talchai C, et al., Nat Genet. 2012 44(4):406-12), This recent paper demonstrates that a similar technique also works in human intestinal cells.

The gene of interest, FOXO1, is indeed present in human gut endocrine progenitor and serotonin-producing cells. In order to determine in FOXO1 inhibition could induce the formation of insulin-secreting cells, Accili and others used human induced pluripotent stem cells (iPSCs) and small “gut organoids,” which are small balls of gut tissue that grow in culture.

Inhibition of FOXO1 by either introducing a mutant version of the gene that encoded a protein that soaked up all the wild-type protein or by using viruses that forced the expression of a small RNA that prevented the expression of the FOXO1 gene caused loss of FOXO1 activity. FOXO1 inhibition promoted the generation of insulin-positive cells within the gut organoids that express all the genes and proteins normally found in mature pancreatic β-cells. These transdifferentiated cells also released “C-peptide,” which is a byproduct of insulin production, in response to drugs that drive insulin secretion (insulin secretagogues). Furthermore, these cultured insulin-secreting cells and survive when transplanted into mice where they continue to secrete insulin in response to increased blood sugar concentrations.

The findings of Accili and his colleagues provide some evidence that gut-targeted FOXO1 inhibition or transplantation of cultured gut organoids made from iPSCs could serve as a source of insulin-producing cells to treat human diabetes.

This is a remarkable piece of research, but there is one thing that troubles me about it. If the patient’s immune system has been sensitized to beta cells, making new beta cells will simply give the immune system something else to attack. It seems to me that retraining to immune system needs to be done first before replacement of the beta cells can ever hope to succeed.

Neurons Made from Induced Pluripotent Stem Cells Stably Integrate into the Brain


Jens Schwamborn and Kathrin Hemmer from the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg have shown that implanted neurons made from induced pluripotent stem cells show long-term stability in the brain.

Induced pluripotent stem cells (iPSCs) are made from mature adult cells by means of genetic engineering and cell culture techniques. These cells have embryonic stem cell-like capacities and can, potentially differentiate into any adult cell type. Because neurons made from iPSCs have sometimes not shown instability, the ability of neurons derived from iPSCs to stably integrate into brain has been questioned.

Schwamborn and Hemmer showed that six months after implantation, their iPSCs-derived neurons had become fully functionally integrated into the brain. This successful integration of iPSC-derived neurons into lastingly stable implants raises hope for future therapies that will replace sick neurons with healthy ones in the brains of patients with Parkinson’s disease, Alzheimer’s disease and Huntington’s chorea, for example. This work was published in the current issue of Stem Cell Reports.

The LCSB research group hopes to bring cell replacement therapy to maturity as a treatment for neurodegenerative diseases. The replacement of sick and/or dead neurons in the brain could one day cure disorders such as Parkinson’s disease. However, devising a successful therapy in human is a long, arduous process, and for good reasons. “Successes in human therapy are still a long way off, but I am sure successful cell replacement therapies will exist in future. Our research results have taken us a step further in this direction,” declared Schwamborn.

In their latest tests, the LCSB research group, in collaboration with colleagues from the Max Planck Institute and the University Hospital Münster and the University of Bielefeld, made stable neuronal implants in the brain from neurons that were derived from reprogrammed skin cells. They used a newer technique in which the neurons were produced from neural stem cells (NSCs). These NSCs or induced neural stem cells (iNSCs) had, in turn been made from iPSCs that were made from the host animal’s own skin cells, which considerably improves the compatibility of the implanted cells. Mice who received the neuronal implants showed no adverse side effects even six months after implantation. The new neurons were implanted into the hippocampus and cortex regions of the brain. Implanted neurons were fully integrated into the complex network of the brain and they exhibited normal activity and were connected to the original brain cells via newly formed connections known as synapses, which are the contact points between nerve cells.

These tests demonstrate that stem cells researchers are continuing to get a better handle on how to use cells derived from something other than human embryos in order to successfully replace damaged or dead tissue. “Building upon the current insights, we will now be looking specifically at the type of neurons that die off in the brain of Parkinson’s patients – namely the dopamine-producing neurons,” Schwamborn reports.

In future experiments, implanted neurons could provide the neurotransmitter dopamine (which is lacking in patients with Parkinson’s disease) directly into the patient’s brain and transport it to the appropriate sites. Such a result would herald an actual cure for the disease rather than a short-term fix. The first trials in mice are in progress at the LCSB laboratories on the university campus Belval.

Thymosin beta4-Overexpressing Cells Heal Heart After a Heart Attack


Thymosin beta4 is a very highly conserved 43-amino acid peptide that plays a very important role in cell proliferation, migration, and angiogenesis (blood vessel production). Experiments with thymosin beta4 in laboratory animals that have had a heart attack have shown that treatment with thymosin beta4 can reduce cell death in the heart and reduce the size of the infarct, while increasing heart function (see Hannappel E, et al., Arch Biochem Biophys 240 (1985): 236-241; Bock-Marquette, et al., Nature 432 (2007): 466-472; Srivastava D, et al., Ann NY Acad Sci 1112 (2007): 161-170; Grant DS et al., Angiogenesis 3 (1999): 125-135). Also, knocking down thymosin beta4 in endothelial progenitor cells (cells that make blood vessels) prevents these cells from healing the heart after a heart attack (Hinkel, et al., Circulation 117 (2008): 2232-2240).

Thymosin beta4
Thymosin beta4

Given the ability of thymosin beta4 to heal the heart, Dinender Singla and colleagues at the University of Central Florida have engineered embryonic stem cells to express thymosin beta4 and used them to treat laboratory animals that have suffered a heart attack. The results were truly tremendous.

Singla and his team genetically engineered mouse embryonic stem cells to express either red fluorescent protein or red fluorescent protein and thymosin beta4. In culture, those cells that expressed thymosin beta4 showed much more efficient differentiation into heart muscle cells (3-5 times greater).

Effect of Tβ4 Expression on ES Cell Differentiation. A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.
Effect of Tβ4 Expression on ES Cell Differentiation.
A. Fluorescent microscopy of EBs derived from RFP-ES and Tβ4-ES cells. At D12 EBs were stained with anti- sarcomeric α-actin (S-actin) (green) and counterstained with DAPI for nuclear visualization (blue). The lower panel shows S-actin staining in a beating area (square box) in the EBs derived from Tβ4-ES cells. Scale = 200µm. B. Percentage of beating EBs during cardiac myocyte differentiation. Spontaneously beating EBs were examined and counted under a light microscope at D9, 12 and 15. C. Real-time PCR analysis of gene expression of GATA-4, Mef2c and Tbx6 at D12. Data are represented as mean ± SEM, *p< 0.05; vs. RFP ESCs.

Next, they gave laboratory mice heart attacks and implanted these cells into the heart. Those mice that received no cells had bucket loads of cell death. Those mice who received embryonic stem cells that did not express thymosin beta4 showed a decrease in cell death 2 weeks after the heart attack. However those mice that received the embryonic stem cells that expressed thymosin beta4 showed a third of the cell death found in the control mice. The same applied to the amount of scarring in the hearts. Animals treated with embryonic stem cells (ESCs) that did not express thymosin beta4 had about half the scarring of the control mice that received no cells, but the hearts treated with thymosin beta4-expressing ESCs showed about a third of the scarring.

Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart. A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. C. Histogram illustrates quantitative MMP-9 expression. #p<0.05 vs sham, *p<0.05 vs. MI. n = 5-7 animals per group.
Transplanted Tβ4-ES Cells Reduce Cardiac Fibrosis in the Infarcted Mouse Heart.
A. Representative photomicrographs of tissue sections stained with Masson’s trichrome at D14 post MI surgery. Scale =100µm. B. Quantitative analysis of interstitial fibrosis for control and experimental groups. #p

When it came to heart function, things were really remarkable. The ESC-treated hearts showed definite improvement over the control animals, but the ESC-thymosin beta4 cells restored heart function so that the hearts worked almost as well as the sham hearts that were never given a heart attack. The fractional shortening was not as high, nor was the end diastolic volume as low, but most of the other functional parameters were close to the sham hearts.

Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart. Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p<0.05 vs. sham, *p<0.05 vs. MI, and $p<0.05 vs. RFP-ESCs. Data set are from n=6-8 animals/group.
Transplanted Tβ4-ES Cells Improve Cardiac Function in the Infarcted Heart.
Echocardiography was performed D14 following MI. A. Raw functional data. Histograms show average quantified measurements of B. left ventricular internal diameter during diastole (LVIDd) C. left ventricular internal diameter during systole (LVIDs) D. fractional shortening FS% E. end diastolic volume (EDV) F. end systolic volume (ESV) G. and ejection fraction EF% at 2 weeks after MI for all treatment groups. #p

Mechanistically, the thymosin beta4 appears to down-regulate PTEN and upregulated the AKT kinase. AKT kinase activation is associated with cell survival and growth. PTEN tends to slow down growth and prevent healing under some conditions.

Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities. Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p<0.01 vs. MI, #p<0.05 vs. sham. n = 4-5 animals per group.
Effects of Tβ4 Expression on Caspase-3, pAkt, and p-PTEN Activities.
Heart homogenates from each group were prepared for ELISA analysis of caspase-3, Akt, and p-PTEN. A. Quantitative analysis of caspase-3, B. p-PTEN, and C. pAkt activity in the hearts following cell transplantation. Data were represented as Mean ± SEM; *p

This suggests that thymosin beta4 expression seems to augment healing in the heart after a heart attack. Such a therapy could potentially be used to treat heart attack patients, however, more animal experiments will need to be done. What is the proper time frame for thymosin beta4 treatment? How many cells should be implanted in order to provide the maximum therapeutic effect. Can such a treatment be provided via intracoronary delivery? Can conditional expression provide a robust enough response to heal the heart? Can other cells, like mesenchymal stem cells to used to deliver the thymosin beta4? Can c-kit cardiac progenitor cells be used to deliver thymosin beta4?

Many questions remain, but hopefully, this remarkable treatment regime can be ramped up to eventually go to clinical trials.