Reliable Generation of Induced Pluripotent Stem Cells From Human Lymphoblastoid Cell Lines

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblastderived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications. STEM CELLS

Growth hormone is permissive for neoplastic colon growth

“…Cells were induced to form hindgut and, ultimately, intestinal organoids. Unlike colon tumor cell lines, intestinal organoids are polarized and comprise several intestinal epithelial subtypes, including enterocytes, goblet cells, and enteroendocrine cells (64) (Fig. S8).…”

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

“…Recombinant Sendai viruses were also shown to be able to efficiently reprogram human hematopoietic cells, including nonlymphocytes to integration-free iPS cells [13,14,38,39]. It is convenient to use premade Sendai viruses (e.g., CytoTune; Life Technologies) from a commercial vendor for reprogramming both hematopoietic and nonhematopoietic cells for laboratory research.…”

Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease

“…Non-integrating iPSC lines were generated from fibroblasts, taken from SMA and matched healthy control subjects, at Cedars-Sinai using the episomal plasmid method of reprogramming as described previously (34). Briefly, iPSC lines were reprogrammed from dermal fibroblasts into virus-free iPSC lines with the Lonza Nucleofector Kit using an episomal plasmid (Addgene) expressing 6 factors: OCT4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA (pCXLE-hOCT3/4-shp53-F, pCXLE-hUL and pCXLE-hSK).…”

“…The iPSCs were grown to near confluence under normal maintenance conditions before the start of the differentiation as per protocols described previously (34,37–39). Briefly, iPSCs were gently lifted by Accutase treatment for 5 min at 37°C.…”