Good Manufacturing Practice‐Grade CD34⁺ Selection from Thawed Cord Blood and Short‐Term Expansion over 3–4 Days as Starting Material for the Generation of Human Leukocyte Antigen Homozygous Induced Pluripotent Stem Cells

Abstract 14

Introduction

On the basis of a funding synergistic consortium by the German BMBF, the cord blood (CB) bank in Düsseldorf was responsible for the selection of human leukocyte antigen (HLA) homozygous (h) donors; contact of the mothers, including re‐consenting; the Good Manufacturing Practice (GMP)‐grade CD34⁺ selection and short‐term expansion of CD34⁺ cells over 3 to 4 days; and certification of an Advanced Therapy Medicinal Products (ATMP)‐raw material according to ATMP‐regulations. Cooperation partners are responsible for the GMP‐grade production of induced pluripotent stem cells (iPSC) and analysis of genetic stability.

Objective

Among 19,218 licensed Düsseldorf cord blood units (PEI.H.00383.01.1, PEI.H.00383.02.1), we were able to identify 139 potential HLAh donors with the most frequent 10 German haplotypes; 100% of the donors were contacted, and 50% were available and consent was obtained. Sequencing for HLA‐A, ‐B, ‐C, ‐DR, ‐DQ, and ‐DP was performed.

Methods

Thawing and washing of the Düsseldorf cord blood was performed in the presence of Volulyte/HSA (Sepax2), CD34⁺ selection by CliniMACS‐system (Miltenyi) applying GMP‐reagents only, expansion was performed applying qualified cytokines (IL‐6, IL‐3, TPO, SCF, FLT‐3 ligand, 100 ng/ml each) and media in the GMP‐facility.

Results

To get a purified CD34⁺ product (no contaminating T cells) 3–4 days after ex vivo expansion, we compared days 3 and 4 for the reprogramming experiments (n = 8). The minimal target for total CD34⁺ was greater than or equal to 5 × 10⁵ to 1 × 10⁶ cells, with purity >70% and viability >90% with no contaminating T cells, that could be obtained in n = 8 samples. The purity of CD34⁺ 7AAD determined by flow cytometry was 74.82% ± 11% after 3 days and 92% ± 2% after 4 days. The fold‐change expansion of viable CD34⁺ cells was lower for day 3 compared with day 4 (1 ± 0.26 compared with 4.01 ± 1.51). Because CD3⁺ cells need some time to go into proliferation after thawing, day 4 is the optimal time point. The amount of detectable T cells in the product was, with 0.03% at day 3/4, very low.

Discussion

The data show that at day 4 of expansion a clean CD34⁺ product could be obtained for transduction with plasmids for the generation of HLAh iPSC. Because CD34⁺ cells were also expanded until day 8–10, this method also could be applied for ex vivo expansion of products. As of March 2019, production permission was issued by the local government.