Xeno-Free Reprogramming of Peripheral Blood Mononuclear Erythroblasts on Laminin-521.

Translating human induced pluripotent stem cell (hiPSC)-derived cells and tissues into the clinic requires streamlined and reliable production of clinical-grade hiPSCs. This article describes an entirely animal component-free procedure for the reliable derivation of stable hiPSC lines from donor peripheral blood mononuclear cells (PBMCs) using only autologous patient materials and xeno-free reagents. PBMCs are isolated from a whole blood donation, from which a small amount of patient serum is also generated. The PBMCs are then expanded prior to reprogramming in an animal component-free erythroblast growth medium supplemented with autologous patient serum, thereby eliminating the need for animal serum. After expansion, the erythroblasts are reprogrammed using either cGMP-grade Sendai viral particles (CytoTune™ 2.1 kit) or episomally replicating reprogramming plasmids (Epi5™ kit), both commercially available. Expansion of emerging hiPSCs on a recombinant cGMP-grade human laminin substrate is compatible with a number of xeno-free or chemically defined media (some available as cGMP-grade reagents), such as E8, Nutristem, Stemfit, or mTeSR Plus. hiPSC lines derived using this method display expression of expected surface markers and transcription factors, loss of the reprogramming agent-derived nucleic acids, genetic stability, and the ability to robustly differentiate in vitro to multiple lineages. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolating peripheral blood mononuclear cells using CPT tubes Support Protocol 1: Removal of clotting factors to produce serum from autologous plasma collected in Basic Protocol 1 Basic Protocol 2: PBMC expansion in an animal-free erythroblast expansion medium containing autologous serum Basic Protocol 3: Reprogramming of expanded PBMCs with Sendai viral reprogramming particles Alternate Protocol: Reprogramming of expanded PBMCs with episomal plasmids Basic Protocol 4: Picking, expanding, and cryopreserving hiPSC clones Support Protocol 2: Testing Sendai virus kit-reprogrammed hiPSC for absence of Sendai viral RNA Support Protocol 3: Testing Epi5 kit-reprogrammed hiPSC for absence of episomal plasmid DNA Support Protocol 4: Assessing the undifferentiated state of human pluripotent stem cell cultures by multi-color immunofluorescent staining and confocal imaging Support Protocol 5: Coating plates with extracellular matrices to support hiPSC attachment and expansion.

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.

Sexual dimorphism in cancer.

The incidence of many types of cancer arising in organs with non-reproductive functions is significantly higher in male populations than in female populations, with associated differences in survival. Occupational and/or behavioural factors are well-known underlying determinants. However, cellular and molecular differences between the two sexes are also likely to be important. In this Opinion article, we focus on the complex interplay that sex hormones and sex chromosomes can have in intrinsic control of cancer-initiating cell populations, the tumour microenvironment and systemic determinants of cancer development, such as the immune system and metabolism. A better appreciation of these differences between the two sexes could be of substantial value for cancer prevention as well as treatment.