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Effects of miRNA-30a 5p knock down in bone marrow CD71-positive erythroid precursor cells (imBMEP-A)

ABSTRACT: Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies were described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent and hematopoietic stem cells. With imBMEP-A, we have previously generated the first erythroid cell line directly derived from reticulocyte progenitors. However, the translation of cultured RBCs into clinical use is currently impeded by the inability to scale up to clinically meaningful doses primarily due to impaired in vitro enucleation of RBC progenitor cell lines in general. To identify factors contributing to the inefficient enucleation in immortalized RBC progenitor cell lines, we conducted a proteome analysis of the imBMEP cell line in comparison to hematopoietic stem cells (HSCs). For a detailed description of the cell culture condition, please see linked publication.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Erythrocyte, Epithelial Cell, Electrically Active Cell, Fibroblast, Embryonic Stem Cell, Blood

SUBMITTER: Kristin Schubert

LAB HEAD: Kristin Schubert

PROVIDER: PXD071954 | Pride | 2025-12-15

REPOSITORIES: Pride

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Publications

Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.

Thiel Jessica J Sürün Duran D Brändle Desiree C DC Teichert Madeleine M Künzel Stephan R SR Friedrich Ulrike U Dahl Andreas A Schubert Kristin K Rzagalinski Ignacy I Shevchenko Andrej A Traikov Sofia S Mirtschink Peter P Wagenführ Lisa L Buchholz Frank F Hölig Kristina K Tonn Torsten T Kronstein-Wiedemann Romy R

Stem cell reviews and reports 20250826 8

In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to imp ...[more]

PMID: 40856920

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Project description:Epigenetic memory in induced pluripotent stem cells (iPSCs), with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, iPSCs from human CD34+ hematopoietic stem cells (HSCs) might be more suitable for hematopoietic differentiation than commonly used fibroblast-derived iPSCs. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). Although genome-wide DNA methylation profiling at all promoter regions demonstrates an epigenetic memory of iPSCs with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. All human iPSC lines showed terminal maturation into normoblasts and enucleated RBCs, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the CD34+ HSC-derived iPSCs. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+ iPSCs and NSC iPSCs, which confirms their comparable erythroid differentiation potential. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300ng of total RNA per sample was used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating the biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. 8 samples were analyzed: CD34 1, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 1, 1 replicate CD34 2, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 2, 1 replicate CD34 OSiPS 1, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSKMiPS 1, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate CD34 OSiPS 2, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate CD34 OSKMiPS 2, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate H1, Human H1 embryonic stem cell (ESC), 1 replicate H9, Human H9 embryonic stem cell (ESC), 1 replicate

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