Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.     

Tracking and Predicting Human Somatic Cell Reprogramming Using Nuclear Characteristics

Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) generates valuable resources for disease modeling, toxicology, cell therapy, and regenerative medicine. However, the reprogramming process can be stochastic and inefficient, creating many partially reprogrammed intermediates and non-reprogrammed cells in addition to fully reprogrammed iPSCs. Much of the work to identify, evaluate, and enrich for iPSCs during reprogramming relies on methods that fix, destroy, or singularize cell cultures, thereby disrupting each cell’s microenvironment. Here, we develop a micropatterned substrate that allows for dynamic live-cell microscopy of hundreds of cell subpopulations undergoing reprogramming while preserving many of the biophysical and biochemical cues within the cells’ microenvironment. On this substrate, we were able to both watch and physically confine cells into discrete islands during the reprogramming of human somatic cells from skin biopsies and blood draws obtained from healthy donors. Using high-content analysis, we identified a combination of eight nuclear characteristics that can be used to generate a computational model to predict the progression of reprogramming and distinguish partially reprogrammed cells from those that are fully reprogrammed. This approach to track reprogramming in situ using micropatterned substrates could aid in biomanufacturing of therapeutically relevant iPSCs and be used to elucidate multiscale cellular changes (cell-cell interactions as well as subcellular changes) that accompany human cell fate transitions.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS)cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs)are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cells reprogrammed from somatic cells using lentivirai expressed reprogramming factors.In our study,iPS cells expressed common pluripotency markers,displayed human embryonic stern cells(hESCs)morphology and unmethylated promoters of NANOG and OCT4.These data demonstrate that SNL feeder cells can support the derivation and maintenance of human iPS cells.     

Induced Pluripotency for Translational Research

The advent of induced pluripotent stem cells （iPSCs） has revolutionized the concept of cellular reprogramming and potentially will solve the immunological compatibility issues that have so far hindered the application of human pluripotent stem cells in regenerative medicine. Recent findings showed that pluripotency is defined by a state of balanced lineage potency, which can be artificially instated through various procedures, including the conventional Yamanaka strategy. As a type of pluripotent stem cell, iPSCs are subject to the usual concerns over purity of differen- tiated derivatives and risks of tumor formation when used for cell-based therapy, though they pro- vide certain advantages in translational research, especially in the areas of personalized medicine, disease modeling and drug screening, iPSC-based technology, human embryonic stem cells （hESCs） and direct lineage conversion each will play distinct roles in specific aspects of translational medi- cine, and continue yielding surprises for scientists and the public.     

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

Application of reprogrammed patient cells to investigate the etiology of neurological and psychiatric disorders

Cellular reprogramming allows for the de novo generation of human neurons and glial cells from patients with neurological and psychiatric disorders.Crucially,this technology preserves the genome of the...     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

The genomic stability of induced pluripotent stem cells

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells of human patients with defined factors, hold promise for regenerative medicine because they can provide a renewable source of autologous cells for cell therapy without the concern for immune rejection. In addition, iPSCs provide a unique opportunity to model human diseases with complex genetic traits, and a panel of human diseases have been successfully modeled in vitro by patient-specific iPSCs. Despite these progresses, recent studies have raised the concern for genetic and epigenetic abnormalities of iPSCs that could contribute to the immunogenicity of some cells differentiated from iPSCs. The oncogenic potential of iPSCs is further underscored by the findings that the critical tumor suppressor p53, known as the guardian of the genome, suppresses induced pluripotency. Therefore, the clinic application of iPSCs will require the optimization of the reprogramming technology to minimize the genetic and epigenetic abnormalities associated with induced pluripotency.