Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells

Heshusius, Steven; Heideveld, Esther; Burger, Patrick; Thiel-Valkhof, Marijke; Sellink, Erica; Varga, Eszter; Ovchynnikova, Elina; Visser, A.S.; Martens, Joost H.A.; Lindern, Marieke von; Akker, Emile van den

doi:10.1182/bloodadvances.2019000689

Cited by 77 publications

(169 citation statements)

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“…The proliferation rate remains variable depending on the origin of HSC, even if there are no proteomic differences between cells from cord blood or peripheral blood [17][18][19]. Recently, the development of a Good Manufacturing Practice (GMP)-grade medium allowed the increased production of enucleated erythrocytes containing adult hemoglobin [20]. Based on Cellquin medium, GPM-grade medium includes growth factors, hormone combinations, and human protein-based media [21].…”

Section: Hematopoietic Stem Cellsmentioning

confidence: 99%

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An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

Deleschaux

Moras

Lefevre

et al. 2020

IJMS

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Human erythropoiesis is a complex process leading to the production of mature, enucleated erythrocytes (RBCs). It occurs mainly at bone marrow (BM), where hematopoietic stem cells (HSCs) are engaged in the early erythroid differentiation to commit into erythroid progenitor cells (burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E)). Then, during the terminal differentiation, several erythropoietin-induced signaling pathways trigger the differentiation of CFU-E on successive stages from pro-erythroblast to reticulocytes. The latter are released into the circulation, finalizing their maturation into functional RBCs. This process is finely regulated by the physiological environment including the erythroblast-macrophage interaction in the erythroblastic island (EBI). Several human diseases have been associated with ineffective erythropoiesis, either by a defective or an excessive production of RBCs, as well as an increase or a hemoglobinization defect. Fully understanding the production of mature red blood cells is crucial for the comprehension of erythroid pathologies as well as to the field of transfusion. Many experimental approaches have been carried out to achieve a complete differentiation in vitro to produce functional biconcave mature RBCs. However, the various protocols usually fail to achieve enough quantities of completely mature RBCs. In this review, we focus on the evolution of erythropoiesis studies over the years, taking special interest in efforts that were made to include the microenvironment and erythroblastic islands paradigm. These more physiological approaches will contribute to a deeper comprehension of erythropoiesis, improve the treatment of dyserythropoietic disorders, and break through the barriers in massive RBCs production for transfusion.

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Section: Hematopoietic Stem Cellsmentioning

confidence: 99%

“…Recently, the development of a Good Manufacturing Practice (GMP)-grade medium allowed the increased production of enucleated erythrocytes containing adult hemoglobin [20]. Based on Cellquin medium, GPM-grade medium includes growth factors, hormone combinations, and human proteinbased media [21].…”

Section: Hematopoietic Stem Cellsmentioning

confidence: 99%

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

Deleschaux

Moras

Lefevre

et al. 2020

IJMS

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“…Data on erythroblast differentiation were retrieved from our previous study (Heshusius et al, 2019). circRNA identification was performed with previously established circRNA detection pipeline (Nicolet et al, 2018).…”

Section: Circrna Identification and Analysismentioning

confidence: 99%

“…GSE124363 (Heshusius et al, 2019). Ribosome footprinting datasets of platelets and erythroblasts were retrieved from GEO with accession number: GSE85864 (Mills et al, 2016).…”

Section: Data Retrieval and Depositionmentioning

confidence: 99%

“…It is made The copyright holder for this preprint this version posted October 22, 2020. ; https://doi.org/10.1101/2020.10.22.348276 doi: bioRxiv preprint circRNA / decreased mRNA (group 2, red), increased circRNA / equal mRNA (group 3, purple), increased circRNA / increased mRNA (group 4, orange), equal circRNA (group 5, grey), decreased circRNA / increased mRNA (group 6, grey). (D) Overlap in circRNA expression between in vitro RBC and ex vivo RBC (data from (Doss et al, 2015;Heshusius et al, 2019;Nicolet et al, 2018); left panel; Figure 1), and in vitro megakaryocytes and ex vivo platelets ( (Kissopoulou et al, 2013;Nicolet et al, 2018); right panel). (E) Overlap between circRNA in differentiated cells and mRNA in progenitor cells, for erythroid (left panel) and megakaryoid cells (right panel).…”

Section: Author Contributionsmentioning

confidence: 99%

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Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA-counterpart in terminal hematopoiesis

Nicolet

Jansen

Heideveld

et al. 2020

Preprint

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Each day, about 10^12 erythrocytes and platelets are released into the blood stream. This substantial output from hematopoietic stem cells is tightly regulated by transcription factors and epigenetic modifications. Whether and how non-coding RNAs such as circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not well understood. We recently reported a circRNA expression map of hematopoietic cells, revealing that erythrocytes and platelets contain the highest levels and numbers of circRNAs. Here, we provide the first detailed and comprehensive analysis of circRNA expression during red blood cell and megakaryocyte differentiation. CircRNA expression significantly increased during erythroid precursor differentiation into red blood cells and in differentiating megakaryocytes, in particular upon enucleation. To determine if circRNAs can modulate hematopoietic differentiation, we compared the expression levels of circRNAs and mRNAs with that of ribosomal foot printing read. This multi-omics approach revealed that only 20 out of 748 (2.6%) circRNAs associated with translation regulation of their mRNA counterparts. Furthermore, irrespective of the thousands of identified putative open reading frames in circRNAs, deep ribosome-footprinting sequencing and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs in erythroblasts, megakaryocytes and platelets. In conclusion, circRNAs in platelets and red blood cells are highly abundant and alter their expression profile during differentiation, yet their contribution to regulate cellular processes remains enigmatic.

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An Optimized Human Erythroblast Differentiation System Reveals Cholesterol‐Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

Wang,

Liu,

Zhang

et al. 2024

Advanced Science

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The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. Human cRBCs can be generated from the terminal differentiation of proliferating erythroblasts derived from hematopoietic stem/progenitor cells or erythroid precursors in peripheral blood mononuclear cells. Efficient differentiation and maturation into cRBCs highly depend on replenishing human plasma, which exhibits variable potency across donors or batches and complicates the consistent cRBC production required for clinical translation. Hence, the role of human plasma in erythroblast terminal maturation is investigated and uncovered that 1) a newly developed cell culture basal medium mimicking the metabolic profile of human plasma enhances cell growth and increases cRBC yield upon erythroblast terminal differentiation and 2) LDL‐carried cholesterol, as a substitute for human plasma, is sufficient to support erythroid survival and terminal differentiation ex vivo. Consequently, a chemically‐defined optimized medium (COM) is developed, enabling robust generation of cRBCs from erythroblasts of multiple origins, with improved enucleation efficiency and higher reticulocyte yield, without the need for supplementing human plasma or serum. In addition, the results reveal the crucial role of lipid metabolism during human terminal erythropoiesis.

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Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells

Abstract: Key Points This article provides a defined GMP-grade medium and erythroid culture protocol, resulting in >90% enucleated RBC. This article provides a high-resolution database of RNA expression dynamics at daily intervals during terminal erythroid differentiation.

Cited by 77 publications

References 50 publications

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA-counterpart in terminal hematopoiesis

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol‐Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

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