Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors.

The methodology to create induced pluripotent stem cells (iPSCs) affords the opportunity to generate cells specific to the individual providing the host tissue. However, existing methods of reprogramming as well as the types of source tissue have significant limitations that preclude the ability to generate iPSCs in a scalable manner from a readily available tissue source. We present the first study whereby iPSCs are derived in parallel from multiple donors using episomal, non-integrating, oriP/EBNA1-based plasmids from freshly drawn blood. Specifically, successful reprogramming was demonstrated from a single vial of blood or less using cells expressing the early lineage marker CD34 as well as from unpurified peripheral blood mononuclear cells. From these experiments, we also show that proliferation and cell identity play a role in the number of iPSCs per input cell number. Resulting iPSCs were further characterized and deemed free of transfected DNA, integrated transgene DNA, and lack detectable gene rearrangements such as those within the immunoglobulin heavy chain and T cell receptor loci of more differentiated cell types. Furthermore, additional improvements were made to incorporate completely defined media and matrices in an effort to facilitate a scalable transition for the production of clinic-grade iPSCs.

EBV is necessary for proliferation of dually infected primary effusion lymphoma cells.

Epstein Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are found together in approximately 80% of primary effusion lymphomas (PEL), but their contribution to these cancers is unclear. We found that dominant-negative derivatives of EBNA1 inhibited EBV-positive PEL cells from forming colonies. Those rare PEL cells that proliferated after expression of the dominant-negative derivatives usually expressed these derivatives at low or undetectable levels and continued to maintain their EBV genomes. Those proliferating cells expressing higher levels of the derivatives expressed mutant derivatives that could not bind DNA. These findings indicate that EBV is required to sustain proliferation, as measured by colony formation of dually infected PEL cells. The dominant-negative derivatives of EBNA1 had no effect on the colony-forming ability of five EBV-negative, KSHV-negative hematopoietic cell lines. Surprisingly, they did inhibit the colony-forming ability of EBV-negative, KSHV-positive PEL cells. The small fraction of cells that continued to proliferate expressed only mutants of the EBNA1 derivatives that could no longer bind DNA. These findings indicate that the site-specific DNA-binding activity of EBNA1 or its derivatives when expressed efficiently in EBV-negative, KSHV-positive PEL cells inhibits their colony formation possibly through their binding to the KSHV genome.

Antagonistic control of oxidative stress-induced cell death in Arabidopsis by two related, plant-specific zinc finger proteins.

The most familiar form of plant programmed cell death is the hypersensitive response (HR) associated with successful plant immune responses. HR is preceded by an oxidative burst and the generation of both reactive oxygen intermediates (ROI) and NO. The Arabidopsis LSD1 gene encodes a negative regulator of plant programmed cell death that meets several criteria for a regulator of processes relevant to ROI management during pathogen responses. Here we demonstrate that a highly conserved LSD1 paralogue, LOL1, acts as a positive regulator of cell death. Manipulation of LOL1 expression alters both the superoxide-dependent, runaway cell death phenotype of lsd1 plants and the normal HR. We also show that LSD1 and LOL1 have antagonistic effects on copper-zinc superoxide dismutase accumulation, consistent with functions in cell death control via maintenance of ROI homeostasis.

A secondary structure that contains the 5' and 3' splice sites suppresses splicing of duck hepatitis B virus pregenomic RNA.

Pregenomic RNA (pgRNA) plays two major roles in the hepadnavirus life cycle. It is the mRNA for two proteins required for DNA replication, C and P, and it is the template for reverse transcription. pgRNA is a terminally redundant transcript whose synthesis does not involve RNA splicing. For duck hepatitis B virus (DHBV), a spliced RNA is derived from pgRNA by removal of a single intron. The mechanism for the simultaneous cytoplasmic accumulation of unspliced (pgRNA) and spliced RNA was not known. We found that mutations within two regions of the DHBV genome reduced the level of pgRNA while increasing the level of spliced RNA. One region is near the 5' end of pgRNA (region A), while the second is near the middle of pgRNA (region B). Inspection of the DHBV nucleotide sequence indicated that region A could base pair with region B. The 5' and 3' splice sites of the intron of the spliced RNA are within regions A and B, respectively. Substitutions that disrupted the predicted base pairing reduced the accumulation of pgRNA and increased the accumulation of spliced RNA. Restoration of base pairing, albeit mutant in sequence, resulted in restoration of pgRNA accumulation with a decrease in the level of spliced RNA. Our data are consistent with a model in which splicing of the pgRNA is suppressed by a secondary structure between regions A and B that occludes the splicing machinery from modifying pgRNA.