Derivation of Induced Pluripotent Stem Cells from Human Peripheral Blood T Lymphocytes

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (“TiPS”) retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages

Small-Scale Telomere Repeat Sequence Content Assay Using Pyrophosphorolysis Coupled with ATP Detection

Studies of telomere length have been carried out in diverse areas of research. However, current methods to measure telomeres are cumbersome and not amenable to high-throughput analyses. Using a coupled pyrophosphorolysis/trans-phosphorylation reaction, we have developed a novel assay to quantitate telomere sequence content in a single tube or 96-well format. The method uses a telomere-specific oligonucleotide probe to sample nanogram quantities of DNA without PCR amplification. Polymerase and kinase enzymes drive the production of ATP, which is then monitored with a luciferase enzyme reporter system. Using this approach, we demonstrated that the luminescent output was linear across a 100-fold range of DNA input, and the assay was sensitive to 0.4–1 ng DNA. A control probe reaction and a DNA quantitation reaction were also designed using the same pyrophosphorolysis technology to correct for background activity and normalize the signal against variations in DNA input, respectively. Finally, we show that the normalized luminescent signal generated by this new method is highly correlated to the telomere restriction fragment length for six human cell lines

Embryonic Stem Cell-Derived Glial Precursors: A Source of Myelinating Transplants

Self-renewing, totipotent embryonic stem (ES) cells may provide a virtually unlimited donor source for transplantation. A protocol that permits the in vitro generation of precursors for oligodendrocytes and astrocytes from ES cells was devised. Transplantation in a rat model of a human myelin disease shows that these ES cell–derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord. Thus, ES cells can serve as a valuable source of cell type–specific somatic precursors for neural transplantation.</jats:p