Automacs device

Fresh human peripheral blood (n = 3) from healthy donors and from active RA patients (deidentified) was collected after institutional review board (IRB) approval and written consent from donors from The Ohio State University Medical Center, and processed following an earlier-described protocol [18 (link),31 (link)]. In, brief, peripheral blood mononuclear cells were isolated from 40 mL of freshly collected blood using Ficoll-Paque density centrifugation. After removing platelets from the peripheral blood mononuclear cell (PBMC) by washing with PBS, CD14⁺ cells were isolated using an AutoMACS device, and magnetic bead conjugated CD14⁺ antibody and reagents (all from Miltenyi Biotec, San Diego, CA, USA) following an earlier-established protocol [18 (link),19 (link)]. Isolated CD14⁺ cells were used for total RNA was extraction and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis.

Fresh human peripheral blood (n = 6) was collected with an approved IRB and written consent from donors from The Ohio State University Medical Center, Columbus, OH and processed following earlier described protocol.11, 19 In, brief, peripheral blood mononuclear cells were isolated from freshly collected blood using Ficoll‐Paque density centrifugation. CD14⁺ cells were isolated by using an AutoMACS device, CD14⁺ antibody and reagents (all from Miltenyi Biotec, San Diego, CA, USA) following earlier established protocol.11, 20 A part of the CD14⁺ cells was used for RNA extraction, and another part was subjected to protein extraction.

Human umbilical cord blood was collected freshly from The Ohio State University Medical Center with an approved IRB after receiving written informed consent from donors, and all research was performed in accordance with the University guidelines. Collected blood was processed following protocol described earlier^{16 (link)}. In brief, mononuclear cells were collected by Ficoll separation, followed by CD133⁺ cells isolation using AutoMACS device (Miltenyi Biotec, Auburn, CA), and purity of the isolated cells and phenotypes after expansion, were determined by flowcytometry as described later. Isolated CD133⁺ cells were expanded according to the protocol described previously^{16 (link)}. Briefly, eight hundred CD133⁺ cells were seeded onto each well of a 24-well cell culture plate containing glued aminated polyethersulfone (PES) nanofiber scaffold (produced in Hai-Quan Mao’s, lab) in 600 μl of StemSpan SFEM, serum-free expansion medium (Stem Cell Technologies, Vancouver, BC, Canada), with essential supplements. Cells were cultured for 10 days at 37 °C in 5% CO₂ without changing medium. Cell phenotypes were confirmed by flowcytometry before conducting experiments.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy volunteers by using Ficoll–Paque gradient centrifugation. CD45RA+ fractions were obtained after magnetic enrichment using CD45RA microbeads and running “Possel” program in the AutoMACS device (both from Miltenyi Biotec) following manufacturer instructions. As the CD45RA+ fraction is a commonly discarded material from haploidentical transplantation procedures, in some cases, CD45RA+ cells were obtained from the Hematology Service at Hospital La Paz after written informed consent, in accordance with the Declaration of Helsinki and La Paz University Hospital Ethics Committee (ethical code 4917), and this study is part of an approved clinical trial with EudraCT: 2016-003578-42. Buffy coats were obtained from the Transfusions Centre of the Comunidad de Madrid upon institutional review board approval. All donors complied with the requirements regarding quality and safety for the donation, obtaining, storage, distribution and preservation of human cells and tissues under the Spanish specific regulation.
K562mbIL15 and K562mbIL21 cells were kindly provided by Prof. Campana (National University Hospital, Singapore) and Prof. Lee (Nationwide Children’s Hospital, Ohio, EEUU), respectively and irradiated with 100 Gy before coculture.