Human lymphoblastoid B-cell lines reprogrammed to EBV-free induced pluripotent stem cells

Abstract

Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations.We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hema- IntroductionPatient-specific induced pluripotent stem cells (iPSCs) can serve as useful models for understanding the etiology of disease and facilitating the development of novel therapeutic interventions. 1 B cells represent a larger fraction of the peripheral blood mononuclear cell population (ϳ 20%) and can be transformed in vitro by Epstein-Barr virus (EBV) to generate lymphoblastoid cell lines (LCLs) using as little as 0.5 mL blood, 2 creating an unlimited proliferative source of cells for reprogramming trials. LCLs are a precious resource for immunologic, epidemiologic, and rare disease studies. A number of facilities manage collections of LCLs available internationally to researchers. 2 Thus, generating iPSCs from LCLs offers the advantage of working with minimal amounts of blood from living donors as well as frozen LCL collections banked worldwide.The capability to reprogram terminally differentiated cells depends on the inherent physiologic plasticity of the cell type. B lymphocytes can transdifferentiate to macrophages 3,4 or hematopoietic precursor cells (HPCs) after down-regulation of Pax5 expression. 4 Murine B cells have been reprogrammed to iPSCs via viral transduction of reprogramming factors with 5 and without Pax5 inhibition. 6 Generating iPSCs via nonviral, nonintegrating methods is appealing to generate clinically useful iPSCs. Recently, iPSCs have been generated by delivering the reprogramming factors via oriP/EBNA-1-based plasmids in fibroblasts and peripheral blood CD34 ϩ cells. 7,8 The inherent plasticity of B cells, their receptivity to oriP/EBNA-1 plasmids, ease of generating LCLs, and availability of banked LCL collections inspired our efforts to reprogram LCLs using oriP/EBNA-1-based vectors.LCL-derived iPSCs (LCL-iPSCs) demonstrated the characteristics of pluripotent stem cells, a normal karyotype, the genetic identity, and IgGH signature of the parental LCLs and lost expression of the episomal reprogramming genes as well as viral genes, leading to self-sustained LCL-iPSCs essentially free of exogenous reprogramming and viral elements. MethodsDetailed methods are included in supplemental Methods (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). All animal experiments were conducted according to relevant national and international guideline...

Will Brain Cells Derived From Induced Pluripotent Stem Cells or Directly Converted From Somatic Cells (iNs) Be Useful for Schizophrenia Research?

“…43 Even though the parental cell lines expressed viral EBNA1 and other Epstein-Barr virus-related elements, these were not detectable in LCL-iPSCs. LCLs harbor artifacts such as point mutations and CNVs, but lines with mutations confirmed in nontransformed cells could provide a source for known high-risk genetic factors such as specific CNVs.…”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…Blood sample collection and storage are among the routine procedures in hospitals and clinics. Therefore, significant efforts have been made to develop efficient and safe procedures for reprogramming various types of blood cells (Table 1) [10,11,[13][14][15][16][17][18][19][20][21]. Umbilical cord blood (UCB) cell banks provide another rich source for cells with diverse HLA types.…”

“…These successes allowed efficient generation of patient-specific iPSCs with intact genomes from convenient cell sources. More recently, it has been shown that EBV-immortalized lymphoblast cells (established originally from blood MNCs) can also be reprogrammed to EBV genome-free iPSCs [20,21]. EBV-immortalized lymphoblasts may have additional somatic mutations (see below for discussion on somatic mutations found in iPSCs) and therefore are not preferred choice as a source of iPSCs to be used in cell therapy.…”

iPSCs for personalized medicine: what will it take for Africa?

“…iPSCs for dissecting complex disease mechanisms and studying genetics Current models for dissecting the genetics underlying complex diseases rely on genome wide association studies (GWAS) performed on human samples from populations with or without the disease of interest [3,6]. Typically, these GWAS involve sample collections of blood, from which DNA is extracted for SNP genotyping analysis.…”