Patient-specific Cell Lines Research Articles

High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

SummaryCRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

Taming the lion: the challenge of immunity in regenerative medicine.

Taming the lion: the challenge of immunity in regenerative medicine.

Patient-Specific iPSC-Derived RPE for Modeling of Retinal Diseases.

Inherited retinal diseases, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world. Currently, treatments for these conditions are limited. Recently, considerable attention has been given to the possibility of using patient-specific induced pluripotent stem cells (iPSCs) as a treatment for these conditions. iPSCs reprogrammed from adult somatic cells offer the possibility of generating patient-specific cell lines in vitro. In this review, we will discuss the current literature pertaining to iPSC modeling of retinal disease, gene therapy of iPSC-derived retinal pigmented epithelium (RPE) cells, and retinal transplantation. We will focus on the use of iPSCs created from patients with inherited eye diseases for testing the efficacy of gene or drug-based therapies, elucidating previously unknown mechanisms and pathways of disease, and as a source of autologous cells for cell replacement.

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient's diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Gene Therapy in Patient-specific Stem Cell Lines and a Preclinical Model of Retinitis Pigmentosa With Membrane Frizzled-related Protein Defects

Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice--an established preclinical model of RP--and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.

Protein signatures of oxidative stress response in a patient specific cell line model for autism

BackgroundKnown genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress.MethodsProtein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways.ResultsThe protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects’ and the ASD patients’ set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress.ConclusionsRedox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.

Human therapeutic cloning, pitfalls and lack luster because of rapid developments in induced pluripotent stem cell technology

Abstract Background: Therapeutic cloning is the combination of somatic cell nuclear transfer (SCNT) and embryonic stem cell (ES) techniques to create specific ES cells that match those of a patient. Because ES cells derived by nuclear transfer (SCNT ES cells) are genetically identical to the donor, it will not generate rejection by the host’s immune system and thus therapeutically may be more acceptable. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cell artificially derived from an adult somatic cell by inducing a forced expression of a set of specific pluripotent genes. In the past few years, rapid progress in reprogramming and iPS technology has been made, and it seems to shadow any progress made in SCNT programs. Objective: This review compares the application perspective of SCNT with that of iPS in regenerative medicine. Methods:We conducted a literature search using the MEDLINE (PubMed), Wiley InterScience, Springer, EBSCO, and Annual Reviews databases using the keywords “iPS”, “ES”, “SCNT” “induced pluripotent stem cells”, “embryonic stem cells”, “therapeutic cloning”, “regenerative medicine”, and “somatic cell nuclear transfer”. Only articles published in English were included in this review. Results: These two methods both have advantages and disadvantages. Nevertheless, by using SCNT to generate patient-specific cell lines, it eliminates complications by avoiding the use of viral vectors during iPS generation. Success in in vitro matured eggs from aged women and even differentiation of oocytes from germ stem cells will further enhance the application of SCNT in regenerative medicine. Conclusion: Human SCNT may be an appropriate mean of generating patient stem cell lines for clinical therapy in the near future.

Human iPSC Models: A Platform for Investigating Neurodevelopmental Diseases

Many human diseases arise as the result of DNA mutations in the patient’s genome. The neurodevelopmental diseases of early childhood have proven difficult to model due to lack of access to embryonic tissue and ethical concerns. Federal restrictions on the use of embryonic material also preclude studying some stages of neurodevelopmental disease. The onset of illness in utero or early childhood is frequently preceded by normal development of critical milestones. Recent work has led to methodologies to transform somatic cells to embryoniclike stem cells using four exogenous transcription factors. With this approach, it is now possible to validate the use of human induced pluripotent stem cells (hiPSCs) to model aspects of neurodevelopmental diseases using a patient’s donated cells or genome editing of hiPSC cells to contain known disease mutations. The reprogramming of somatic cells to hiPSC requires dedifferentiation and resetting of epigenetic signatures in the genome. The newest approaches are evaluating propagating the cells in three dimensions on artificial matrices to recapitulate regional neural cyto-architecture within the brain. Newer genome editing techniques that rely on site-specific sequence recognition by synthetic enzymes can be used to generate hiPSC neurodevelopmental disease models. A hiPSC disease model has several advantages, the patient’s own cells may be transduced to provide the investigative cell model and compared to other patient’s cells with the same disease. Additionally, a hiPSC model addresses some of the concerns about gene engineered animal models accurately recapitulating human disease since the model context is a patient-specific human cell line. Here we review the emerging use of hiPSC to model neurodevelopmental diseases.

Patient-Specific iPSCs-Based Liver-On-A-Chip

Successful advent of future medicine (e.g., personalized, preventive, predictive, participating medicine) will be strongly bonded to the availability of human-physiology-describing models. The combination of advanced researches in induced personalized stem cells (iPSCs) and the state-of-the-art microengineering is considered to create in-vitro models of organ functions in a near future; the first can generate patient-specific human cell lines, and the latter enables physiologically relevant microenvironments for recapitulating organ-level functions. Here we present a patient-specific iPSC-derived hepatocytes in an integrated microphysiological analysis platform (iMAP). The iMAP for iPSC-derived hepatocyte model is designed to culture the hepatocytes using endothelial-like physical barriers, which separate cell culture chambers and perfusion channels with Peclet number <<1. The physiological architecture supports enhanced hepatic functions of the iPSCs-derived hepatocytes, especially protein synthesis and drug metabolism. Compared to a conventional sandwich culture method, the liver-on-a-chip supports three-dimensional sinusoidal organization, enhanced junction protein and continuously refreshed media around the hepatic tissue to express higher albumin secretion and specifically enhanced drug metabolism (e.g., phenacetin is more metabolized to acetaminophen and N-acetyl-p-benzoquinone imine). Also, iPSCs-derived hepatocytes, by being matured from hepatoblast in the liver-on-a-chip platform, can increase the structural integrity due to avoiding dissociation of polarized hepatocytes and organogenesis-like tissue development in the microenvironment. The iMAP for iPSC-derived hepatocyte model will be tested for patient-specific pharmacokinetics/pharmacodynamics by involving various patient-derived iPSCs, and also other organ models will be integrated to estimate organ-organ interactions, which will provide various human-physiology-describing in-vitro models.

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29hi/CD49fhi/CD24hi markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

Pluripotency-Regulating Networks Provide Basis for Reprogramming

The reprogramming of somatic cells into induced pluripotent stem cells or iPS cells can be achieved by the ectopic expression of defined factors. Patient-specific iPS cell lines can be derived and used for disease modeling, drug and toxicology screening, cellular replacement therapies and basic research. However, reprogramming is slow and inefficient and numerous methods have been described aiming to improve this process. These methods include screening for new genetic factors and chemical compounds, and the engineering of new synthetic factors. Here, we review recent progress made in this field and show how a better understanding of the ES (embryonic stem) cell transcriptional network is important for efficient reprogramming.

Abstract 4155: Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice.

Abstract BACKGROUNDHigh-throughput functional chemosensitivity testing is a potentially effective way to customize personalized chemotherapy treatment. This testing requires low-passage patient-specific cancer cell lines, however, generating such cell lines from patients’ tumors is difficult, due in large part, to contaminating fibroblasts that often overgrow the malignant cells. METHODSWe constructed a series of hypoxanthine phosphoribosyl transferase (hprt) null immunodeficient mice. These include nude hprt-null, SCID hprt-null and NOD-SCID hprt-null mice. During growth of xenografted human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Tumors from patients were implanted and expanded in these mice. Explanted tumors were then grown in tissue culture flasks with selection media (hypoxanthine, aminopterin, thymidine; HAT media) to eliminate the mouse biochemically-defective fibroblasts and produce pure human cancer cell lines. We screened one cell line against the Johns Hopkins Drug Library containing ∼3,100 drugs. Selected potent drugs were used for in vivo xenograft tumor inhibition studies. RESULTSWe successfully established several pancreatic cancer cell lines from patients using the hprt null immunodeficient mice. We performed chemosensitivity profiling on one of the pancreatic cancer cell lines using 3,100 drugs. From the profiling, we selected potent drugs, which are FDA-approved but are not currently used for pancreatic cancer. We chose two drugs that demonstrated greater in vitro chemosensitivity compared to control cell lines. The two drugs were tested against mouse xenografts where both drugs demonstrated superior in vivo efficacy to the sensitive cell line tumors compared to control tumors. CONCLUSIONSChemotherapy for cancer may be personalized using low passage cell lines derived from patients through biochemically selectable mice. Further tests are needed to determine whether in vitro chemosensitivity testing of low-passage patient-specific cancer cell lines can predict clinical patient response. Citation Format: Hirohiko Kamiyama, Sherri Rauenzahn, Joong Sup Shim, Jun O. Liu, Anirban Maitra, James R. Eshleman. Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2013-4155

Personalized Chemotherapy Profiling Using Cancer Cell Lines from Selectable Mice

High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells. We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.

Brief Report: Self-Organizing Neuroepithelium from Human Pluripotent Stem Cells Facilitates Derivation of Photoreceptors

Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling.

The evolving field of induced pluripotency: Recent progress and future challenges

The derivation of patient-specific pluripotent cell lines through the introduction of a few transcription factors into somatic cells has opened new avenues for the study and treatment of human disorders. Induced pluripotent stem cells (iPSCs) and their derivatives offer a unique platform for disease modeling, drug discovery and toxicology, as well as an invaluable source of cells for regenerative therapies. Here, we provide an overview of the various strategies currently available for iPSC generation, highlighting recent advances and discussing some of the challenges faced in harnessing the true potential of iPSCs for biomedical research and therapeutic applications.

Prospects and Challenges of Reprogrammed Cells in Hematology and Oncology

Induced pluripotent stem cells (iPSCs) have emerged as a promising basis for modeling pediatric genetic disorders, allowing the derivation, study, and genetic correction of disease and patient-specific cell lines in vitro. Similar to embryonic stem cells (ESCs), iPSCs are capable of unlimited in vitro expansion and derivation of many cell types, including hematopoietic stem cells (HSCs). These may not only allow large scale screenings to develop therapeutic compounds, but also help to overcome cross-species barriers of genetically engineered animal models, which do not adequately recapitulate the associated human phenotype. Here, we review the current state and emerging developments of iPSC research, which can be exploited as a tool in modeling pediatric hematopoietic disorders and could lead to new clinical applications in gene and cell therapies.

Reduced Production of Mature Neutrophils From Induced Pluripotent Stem Cells Derived From a Severe Congenital Neutropenia Patient with HAX1 Gene Deficiency

Abstract 2402Induced pluripotent stem (iPS) cells are reprogrammed somatic cells with embryonic stem (ES) cell-like characteristics. As iPS cells can be generated from somatic cells of patients with a certain disease, they are expected to be a novel model to study pathogenesis of various diseases. Recently, we established a neutrophil differentiation system from human iPS cells (Morishima T, et al. J Cell Physiol. 2011). In an attempt to apply the system to investigate pathophysiology of neutrophil-affected disorders, we generated iPS cells from a severe congenital neutropenia (SCN) patient with HAX1 gene deficiency.The patient was an 11-year-old boy with severe congenital neutropenia as well as developmental delay and epilepsy. DNA sequence analysis revealed HAX1 gene mutation in exon 2 (Matsubara K, et al. Haematologica. 2007). Four iPS cell lines were generated from skin fibroblasts of the patient by retroviral overexpression of the three or four transcription factors Oct3/4, Sox2, and Klf4, with or without c-Myc. These patient-derived iPS cell lines showed human ES cell like morphology and could be maintained under human ES cell culture condition. They also expressed typical human ES cell markers and were capable of differentiating into the cell lineages and tissues representing three germ layers by teratoma formation in vivo. These cells had normal karyotype and short tandem repeat analysis indicated that they were derived from parental skin fibroblast. DNA sequencing analysis of the iPS cell lines identified the same mutation carried in the parental skin fibroblasts, thus confirmed that we had established the HAX1 deficiency patient-specific iPS cells (HAX1-iPSCs).Next these HAX1-iPSCs and the healthy-person derived iPS cells were differentiated into neutrophils in vitro using feeder-free culture protocols established in our laboratory. In this culture system, small human iPS cell clumps were cultured on the matrigel-coated dish with recombinant cytokines and without any feeder cells and fetal calf serum. Around day 25 of culture, mature neutrophils were obtained as floating cells. Morphologically, the majority of HAX1-iPSCs-derived cells were classified into myeloblast or promyelocyte stage and there were only a few mature neutrophils. The proportion of mature neutrophils was only less than 10% in HAX1-iPSCs-derived cells whereas more than 40% in normal control. Flow cytometric analysis revealed that the percentage of immature CD34 positive cells was significantly higher and that of myeloid-committed CD11b positive cells was lower in the HAX1-iPSCs-derived cells than normal control. Immunocytochemical analysis for neutrophil specific granules showed that lactoferrin- and gelatinase-positive cells decreased in the HAX1-iPSCs-derived cells compared with normal control, confirming that HAX1-iPSCs-derived cells contained less mature neutrophils than normal control. Apoptosis assay by Annexin V staining revealed that HAX1-iPSCs-derived cells showed higher percentage of Annexin V-positive cells compared with normal control.Overall, these HAX1 deficiency patient-specific iPS cell lines recapitulate the hematological phenotype in the patient. These results indicate that patient-derived iPS cells provide us a novel disease model and make a contribution to the understanding of the pathophysiology of the diseases that affect neutrophils. Disclosures:No relevant conflicts of interest to declare.

Efficient induction of functional neurons from adult human fibroblasts

Cellular reprogramming is a rapidly developing technology by which somatic cells are turned into pluripotent stem cells or other somatic cell types through expression of specific combinations of genes. This allows for the generation of patient-specific cell lines that can serve as tools for understanding disease pathogenesis, for drug screens and, potentially, for cell replacement therapies. Several cellular models of neurological disorders based on induced pluripotent cells (iPS cells) have been developed, and iPS-derived neurons are being explored as candidates for transplantation. Recent findings show that neurons can also be induced directly from embryonic and postnatal somatic cells by expression of defined combinations of genes. This conversion does not occur through a pluripotent stem cell stage, which eliminates the risk for tumor formation. Here, we demonstrate for the first time that functional neurons can be generated via direct conversion of fibroblasts also from adult individuals. Thus, this technology is an attractive alternative to iPS cells for generating patient- and disease-specific neurons suitable for disease modeling and autologous transplantation.

Therapeutic cloning by xenotransplanted oocytes, supplemented with species specific reprogramming factors

For therapeutic medication, a lot of work has been done regarding human embryonic stem (ES) cell lines derivation, but immunorejection is a major dilemma of this cell based therapy. Since long time, derivation of patient matched stem cells have been hoped to overcome this problem. Oocytes after nuclear transfer are the most reliable source for patient matched ES cells derivation, for therapeutic use. In humans, utilization of oocytes for stem cell research raises sensitive logistical and ethical questions; primarily surrounding participation of women as oocyte donors. It has been claimed that therapeutic cloning would lead to commercial exploitation of poor women. On the other hand, the therapeutic promise of embryonic stem cell is so huge that there is a strong incentive to find some alternate sources of human oocytes. Here we propose to utilize the cross species oocytes supplemented with human ES cellular extracts to establish patient specific ES cell lines.

Efficient Derivation of Embryonic Stem Cells from Nuclear Transfer and Parthenogenetic Embryos Derived from Cryopreserved Oocytes

Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.