Generation of Ips Cell-Derived Hematopoietic Progenitor Cells from Patients with Acquired Aplastic Anemia Harboring Copy Number Neutral Loss of Heterozygosity of the Short Arm of Chromosome 6
Authors: Espinoza Luis , Yoshida Yoshinori , Chonabayashi Kazuhisa , Katagiri Takamasa , Zaimoku Yoshitaka , Nakao Shinji 
Published: October 2019

Aplastic anemia (AA) is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. Because of the low number of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow, it has been intrinsically difficult to study AA. Although autoimmunity to HSPCs is considered a key factor responsible for the pathogenesis of this disorder, little is known about the molecular basis of this autoimmunity. We have previously reported that leukocytes lacking HLA class I alleles are frequently detectable in patients with AA and are derived from HSPCs that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH), and thereby escape the cytotoxic T-cell (CTL) attack against HSPCs (Katagiri et al, Blood 2011). Studying HSPCs derived from AA patients with 6pLOH(+) and their 6pLOH(-) counterparts should therefore be useful for identifying specific antigens that are targeted by CTLs in AA. In this study we generated human induced pluripotent stem cells (iPSCs) from monocytes derived from four AA patients with 6pLOH. Our generated iPSCs had normal karyotypes and were positive for human pluripotent stem cell markers such as Oct4 and SSEA4. The proportion of 6pLOH(+) iPSC clones generated from each patient was similar to the proportion 6pLOH(+) cells in the parental monocytes (50%, 100%, 100% and 100%, respectively). Unlike to their parental monocytes and consistent with previous reports, iPSCs poorly expressed class I and class II HLAs. Using an in vitro differentiation system with various hematopoietic cytokines and growth factors, iPSCs were successfully differentiated into CD34+ hematopoietic progenitor cells (iPSC-HPCs). Phenotypic studies revealed that on day 21 of the differentiation process, HPCs expressed CD34 (44.0%), the erythroid cell marker CD235a (10.5%), and CD45 (73.3%) but did not express the lineage-committed marker CD33or CD38, which is a marker expressed by a variety of mature hematopoietic cell types (Figure 1). As expected, both HLA-A alleles were highly expressed by in vitro differentiated HPCs derived from 6pLOH(-) wild type iPSC clones while the one HLA-A allele that was missing on the parental monocytes (HLA-A24) was undetectable in HPCs derived from 6pLOH(+) iPSC clones. Similarly, iPSC-HPCs did not express MHC class I-related chain (MIC)-A/B, the ligands for the NK cell activating receptor NKG2D. To further determine the hematopoietic potential of the iPSC-HPCs, they were cultured in the methylcellulose medium containing various colony stimulating factors. iPSC-HPCs were able to generate various types of colonies including CFU-GEMM (granulocyte, erythrocyte, macrophage, and megakaryocyte) and BFU-E at the plating efficiency of approximately 50%. Our study demonstrated, for the first time, that functional HPCs that lack particular HLA alleles due to 6pLOH can be successfully generated in vitro. The large number of HSCs generated from patients' derived iPSCs may be an excellent tool for searching antigens targeted by CTLs and investigating the pathogenesis of AA and thus developing novel therapeutic approaches.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.