Lucas V. Greder

Cardiac Repair in a Porcine Model of Acute Myocardial Infarction with Human Induced Pluripotent Stem Cell-Derived Cardiovascular Cells

Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Trilineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress, and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair.

In vivo reprogramming of Sox9+ cells in the liver to insulin-secreting ducts

In embryonic development, the pancreas and liver share developmental history up to the stage of bud formation. Therefore, we postulated that direct reprogramming of liver to pancreatic cells can occur when suitable transcription factors are overexpressed. Using a polycistronic vector we misexpress Pdx1, Ngn3, and MafA in the livers of NOD-SCID mice rendered diabetic by treatment with streptozotocin (STZ). The diabetes is relieved long term. Many ectopic duct-like structures appear that express a variety of β-cell markers, including dense core granules visible by electron microscopy (EM). Use of a vector also expressing GFP shows that the ducts persist long after the viral gene expression has ceased, indicating that this is a true irreversible cell reprogramming event. We have recovered the insulin+ cells by cell sorting and shown that they display glucose-sensitive insulin secretion. The early formed insulin+ cells can be seen to coexpress SOX9 and are also labeled in mice lineage labeled for Sox9 expression. SOX9+ cells are normally found associated with small bile ducts in the periportal region, indicating that the duct-like structures arise from this source. This work confirms that developmentally related cells can be reprogrammed by suitable transcription factors and also suggests a unique therapy for diabetes.

Reprogramming of pancreatic exocrine cells towards a beta (β) cell character using Pdx1, Ngn3 and MafA

Pdx1 (pancreatic and duodenal homeobox 1), Ngn3 (neurogenin 3) and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) have been reported to bring about the transdifferentiation of pancreatic exocrine cells to beta (β) cells in vivo. We have investigated the mechanism of this process using a standard in vitro model of pancreatic exocrine cells, the rat AR42j-B13 cell line. We constructed a new adenoviral vector encoding all three genes, called Ad-PNM (adenoviral Pdx1, Ngn3, MafA construct). When introduced into AR42j-B13 cells, Ad-PNM caused a rapid change to a flattened morphology and a cessation of cell division. The expression of exocrine markers is suppressed. Both insulin genes are up-regulated as well as a number of transcription factors normally characteristic of beta cells. At the chromatin level, histone tail modifications of the Pdx1, Ins1 (insulin 1) and Ins2 (insulin 2) gene promoters are shifted in a direction associated with gene activity, and the level of DNA CpG methylation is reduced at the Ins1 promoter. The transformed cells secrete insulin and are capable of relieving diabetes in streptozotocin-treated NOD-SCID (non-obese diabetic severe combined immunodeficiency) mice. However the transformation is not complete. The cells lack expression of several genes important for beta cell function and they do not show glucose-sensitive insulin secretion. We conclude that, for this exocrine cell model, although the transformation is dramatic, the reprogramming is not complete and lacks critical aspects of the beta cell phenotype.

Transgene-Free Disease-Specific Induced Pluripotent Stem Cells from Patients with Type 1 and Type 2 Diabetes

The induced pluripotent stem cell (iPSC) technology enables derivation of patient‐specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient‐specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes‐specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration‐free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification‐free iPSCs (SV‐iPSCs) from patients with diabetes, including an 85‐year‐old individual with T2D. SV‐iPSCs lost the Sendai viral genome and antigens within 8–12 passages while maintaining pluripotency. Genome‐wide transcriptome analysis of SV‐iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b, and p21CIP1. SV‐iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene‐free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes‐associated complications.

Effective Cardiac Myocyte Differentiation of Human Induced Pluripotent Stem Cells Requires VEGF

Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs), which have paved the way for regeneration therapy using patients’ own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines, which inhibit the application of this technology significantly. Here, we demonstrate a novel myocyte differentiation protocol that can yield a significant, high percentage of cardiac myocyte differentiation (>85%) in 2 hiPSC lines, which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts, but also from blood mononuclear cells.

Brief Report: Analysis of Endogenous Oct4 Activation during Induced Pluripotent Stem Cell Reprogramming Using an Inducible Oct4 Lineage Label

The activation of endogenous Oct4 transcription is a key step in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells but until now it has been difficult to analyze this critical event in the reprogramming process. We have generated a transgenic mouse that expresses the tamoxifen‐inducible Cre recombinase MerCreMer under the control of the endogenous Oct4 locus, enabling lineage tracing of Oct4 expression in cells in vivo or in vitro, during either reprogramming or differentiation. Using this novel resource, we have determined the timing and outcome of endogenous Oct4 induction during fibroblast reprogramming. We show that both the initiation of this key reprogramming step and the ability of cells activating endogenous Oct4 expression to complete reprogramming are not influenced by the presence of exogenous c‐Myc, although the overall efficiency of the process is increased by c‐Myc. Oct4 lineage tracing reveals that new reprogramming events continue to initiate over a period of 3 weeks. Furthermore, the analysis of mixed colonies, where only a subset of daughter cells induce endogenous Oct4 expression, indicates the role of unknown, stochastic events in the progression of reprogramming from the initial events to a pluripotent state. Our transgenic mouse model and cells derived from it provide powerful and precise new tools for the study of iPS cell reprogramming mechanisms and have wider implications for the investigation of the role of Oct4 during development. STEM CELLS2012;30:2596–2601

Stable insulin-secreting ducts formed by reprogramming of cells in the liver using a three-gene cocktail and a PPAR agonist

With the long-term aim of developing a new type of therapy for diabetes, we have investigated the reprogramming of liver cells in normal mice toward a pancreatic phenotype using the gene combination Pdx1, Ngn3, MafA. CD1 mice were rendered diabetic with streptozotocin and given a single dose of Ad-PNM, an adenoviral vector containing all three genes. Ad-PNM induced hepatocytes of the liver to produce insulin, and the blood glucose became normalized. But over several weeks, the insulin-positive cells were lost and the blood glucose rose back to diabetic levels. Simultaneous administration of a peroxisome-proliferator-activated receptor agonist, WY14643, caused remission of diabetes at a lower dose of Ad-PNM and also caused the appearance of a population of insulin-secreting ductal structures in the liver. The insulin-positive ducts were stable and were able to relieve diabetes in the long term. We show that the effect of WY14643 is associated with the promotion of cell division of the ductal cells, which may increase their susceptibility to being reprogrammed toward a beta cell fate.

Loss of Oct4 expression during the development of murine embryoid bodies.

We describe the internal organization of murine embryoid bodies (EBs) in terms of the structures and cell types formed as Oct4 expression becomes progressively lost. This is done by making the EBs from iPS cells carrying a novel Oct4 reporter (Oct4-MerCreMer;mTmG) which is inducible, sensitive, and permanent in all cellular progeny. When these EBs are treated with tamoxifen, the Oct4 expressing cells switch from a red to a green fluorescence color, and this is maintained thereafter by all their progeny. We show that there is no specific pattern in which Oct4 is downregulated, rather it appears to be spatially random. Many of the earliest cells to lose Oct4 expression stain positive for markers of visceral endoderm (DAB2, α-fetoprotein (AFP), HNF4). These are randomly located, although if endoderm differentiation is allowed to commence before EB formation then an external layer is formed. This is true both of EBs made from the reporter iPS cells, or from an embryo-derived mouse ES line (R1 cells). Markers of the early body axis, Brachyury (BRA) and FOXA2, usually showed a concentration of positive cells in one region of the EB, but the morphology is not predictable and there are also scattered cells expressing these markers. These patterns are similar in R1 cells. Use of the Oct4 reporter showed a difference between BRA and FOXA2. BRA, which marks the early mesoderm, node and notochord, arises in Oct4 expressing cells on days 3-4. FOXA2, which marks the floor plate of the neural tube and definitive endoderm, as well as the node and notochord, arises at the same time but mostly in cells that have already lost Oct4 expression. Several clumps of cardiomyocytes are visible by days 7-8 of EB development, both in our iPS cells and in R1 cells. Using the Oct4 reporter we show that the cells forming these clumps lose Oct4 expression between days 3 and 5. Overall, our results indicate that EBs recapitulate normal development quite well in terms of the tempo of events and the appearance of specific markers, but they do not resemble embryos in terms of their morphology.

Using Oct4:MerCreMer Lineage Tracing to Monitor Endogenous Oct4 Expression During the Reprogramming of Fibroblasts into Induced Pluripotent Stem Cells (iPSCs)

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) using a combination of defined transcription factors has become one of the most widely used techniques in stem cell biology. A critical, early event in iPSC reprogramming is the induction of the endogenous transcription factor network that maintains pluripotency in iPSCs. Here we describe using a transgenic, conditional Oct4-Cre construct to investigate the spatial and temporal induction of endogenous Oct4 expression during the reprogramming of mouse fibroblasts into iPS cells.