Patient-specific Cell Lines Research Articles

IPS cell technology in regenerative medicine

The promise of treating human genetic and degenerative diseases through the application of pluripotent cell-based tissue engineering and regenerative medicine has come significantly closer to realization since the isolation of human embryonic stem (ES) cells. While the study of ES cells has greatly increased our fundamental understanding of pluripotency, technical and ethical limitations have been seemingly insurmountable impediments to the application of these cells in the clinic. The recent discovery that somatic mammalian cells can be epigenetically reprogrammed to a pluripotent state through the exogenous expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC has yielded a new cell type for potential application in regenerative medicine, the induced pluripotent stem (iPS) cell. Here we discuss how advances in iPS cell technology have led to the generation of patient-specific cell lines that can potentially be used to model human diseases and ultimately act as therapeutic agents.

Induced pluripotent stem (iPS) cells: an up-to-the-minute review

Recent advances in nuclear reprogramming technology allow the transformation of terminally differentiated, adult cells into induced pluripotent stem cells whose phenotype is indistinguishable from that of embryonic stem cells. This leap forward enables the creation of patient-specific pluripotent cell lines that carry disease genotypes. These cell lines could be used both as in vitro models for the study of disease and as potential sources of material for cell replacement therapy. Ultimately, a greater understanding of the process by which cellular identity is shaped and altered may allow the generation of particular cell types for the treatment of degenerative disease.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.

Human Embryonic and Human Induced Pluripotent Stem Cell Lines

Human embryonic stem (ES) cells capture the imagination because after many months of proliferation in vitro, these versatile cells maintain the ability to form potentially any cell type that makes up the body; The proliferative and developmental potential of human ES cells promises an essentially unlimited supply of differentiated cells for basic research, drug discovery, and for transplantation therapies for diseases ranging from heart disease to Parkinson's disease to leukemia. However, human ES cells are derived from preimplantation embryos, making them highly controversial. Using information gleaned from over a decade of research with primate and human ES cells, including the identification of key signaling pathways that promote undifferentiated proliferation and the development of approaches for genetic modification, we have recently derived the first human induced pluripotent stem (iPS) cells, cells with the defining characteristics of ES cells, but derived from somatic, not embryonic material. Human iPS cells thus have all the advantages of ES cells, but avoid the ethical controversy surrounding the destruction of human preimplantation embryos, and in addition allow the derivation of pluripotent cells lines that match the genetic background of a specific individual. For drug discovery and testing, panels of iPS cell lines can now be derived with a genetic composition that accurately reflects the ethnic diversity of a population, and cell lines can be derived from individuals predisposed to specific diseases for the development of new in vitro models of disease. For transplantation therapies based on these cells, patient-specific iPS cell lines largely eliminate the concern of immune rejection. Here we discuss the historical events leading up to the isolation of human ES cells, and how the successful derivation of human iPS cells depended critically on the last decade of human ES cell research.

Induced Pluripotent Stem Cell Lines Derived From Human Somatic Cells

Because the mammalian embryo is regulated by epigenetic rather than genetic events, differentiation is—in principle—reversible. Somatic cell nuclear transfer permits trans-acting factors resident in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. The investigators have found that 4 genes (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that possess the defining features of embryonic stem (ES) cells. Each of 4 induced pluripotent stem (iPS) cells had the typical human ES cell morphology and a normal karyotype after up to 17 months of culture. Each iPS clone expressed telomerase activity and human ES cell-specific cell surface antigens. All of the reprogrammed iPS clones were able to give rise to differentiated derivatives of all 3 primary germ layers. When human newborn foreskin fibroblasts were transduced, each clone consisted of cells having a human ES cell morphology and genes characteristic of human ES cells. When last examined after 14 weeks, all of the iPS clones were proliferating vigorously. Each clone exhibited multilineage differentiation in both embryoid bodies and teratomas. Except for the fact that human iPS cells are not derived from embryos, they meet defining criteria for human ES cells. They should prove helpful when studying the development and function of human tissues. Once technical limitations such as mutation through viral integration are effectively addressed, the cells should find use in transplantation treatments. With the exception of autoimmune diseases, patient-specific iPS cell lines should mostly eliminate concern over immune rejection.

Human embryonic stem cells: preclinical perspectives

Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

A Transcriptional Logic for Nuclear Reprogramming

Limitations on a differentiated cell's pluripotency can be erased by nuclear transfer or by fusion with embryonic stem cells, but attempts to recapitulate this process of nuclear reprogramming by molecular means have failed. In this issue of Cell, Takahashi and Yamanaka (2006) take a rational approach to identifying a suite of embryonic transcription factors whose overexpression restores pluripotency to adult somatic cells.

Designer blood: creating hematopoietic lineages from embryonic stem cells

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

Reactions to the Hwang Scandal

Reactions to the Hwang Scandal

Human adult olfactory neuroepithelial derived progenitors retain telomerase activity and lack apoptotic activity

Olfactory epithelium (OE) contains a population of progenitors responsible for its life-long regenerative capacity. Procedures for the isolation of these progenitors have been established [F.J. Roisen, K.M. Klueber, C.L. Lu, L.M. Hatcher, A. Dozier, C.B. Shields, Adult human olfactory stem cells, Brain Res., 890 (2001) 11–12.] and over 40 patient-specific cell lines from adult postmortem OE and endoscopic biopsy from patients undergoing nasal sinus surgery have been obtained. As these cells emerged in primary cultures, they formed neurospheres (NSFCs). The purpose of the present study was to further characterize these adult human olfactory-derived progenitors. Subcultures of the NSFCs have been passaged nearly 200 times, with a mitotic cycle of 18–20 h. Telomerase activity remains in stem cells; therefore, ELISA was employed to determine the telomerase activity of different lines and passages. Since progenitors undergo low levels of apoptosis, the levels of apoptosis were also examined in these populations. The levels of telomerase and apoptotic activity in 12 NSFC lines remained relatively constant irrespective of donor age, culture duration, or sex. To further study the apoptotic characteristics of the NSFCs, nine different caspases (cysteine proteases) known to be critical in apoptosis were evaluated using gene-microarrays comparing cells from a single line at passages 14, 88, and 183. No increases were found in caspase activity in all passages studied. ELISA confirmed the absence of caspase activity over the entire range of passages. This study further suggests that NSFCs can be obtained and used from patients, irrespective of age, sex, or time in culture without altered viability expanding the potential utility of these cells for autologous transplantation and possible diagnostic testing.