A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

Summary

Modeling erythropoiesis provides a valuable chance to comprehend the biological significance of actors in this process and to evaluate novel therapeutic approaches that may alleviate differentiation defects. Here, we describe a simple and reliable method to efficiently differentiate CD34⁺ hematopoietic stem and progenitor cells ex vivo.

Abstract

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 ± 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

Introduction

Every day about 280 billion red blood cells (RBCs) are produced in the bone marrow to ensure oxygen transport throughout the entire body¹. Erythropoiesis is a finely tuned differentiation process that produces mature, functional red blood cells from hematopoietic stem cells. Many diseases, both acquired and congenital, can affect this process, including myelodysplastic syndrome, aplastic anemia, thalassemia, and congenital dyserythropoietic anemia².

The clinical manifestation of disrupted erythropoiesis is anemia, a major cause of morbidity worldwide. Symptoms of anemia range from fatigue, sho....

Protocol

Research on primary samples was performed in compliance with institutional guidelines (REC reference: 21/EE/0133; IRAS project ID: 283103).

1. Lentivirus production

NOTE Lentiviruses must be produced in a laboratory of biosafety level L2 under sterile conditions. All required PPE for this lab must be worn, including double gloves. Contaminated material is decontaminated with virucide disinfectant prior to disposal in an autoclaved bin. Here, a shRNA s.......

Discussion

Here, we described an efficient and reliable method to induce erythroid differentiation of CD34⁺ hematopoietic stem cells (HSPCs) isolated either from adult bone marrow (BM) or from umbilical cord blood (UCB). This pipeline includes the editing/modification of primary cells to investigate the function of any gene of interest during the erythroid differentiation process. This method has been successfully used to investigate the functional relevance of genes that were not previously linked to erythroid different.......

Materials

Name	Company	Catalog Number	Comments
2x HEPES	Merck	51558
Beads	Thermo Scientific	01-2222-42
CD235a APC Cy7	Biolegend	349115
CD34 PerCP Cy5.5 antibody	BD Biosciences	347222
CD71 PE antibody	Biolegend	334106
DAPI	Merck	D9542
DMEM	Thermo Scientific	41966-029
DNAse	Merck	D4527-200KU
DPX mounting medium	VWR	1.00579.0500
FACSAria Fusion	BD Biosciences	NA
FBS	Merck	F9665
Gelatin	Merck	G1393
Giemsa solution	Abcam	ab150670
HEPES	Merck	H0887-100mL
Human EPO	PeproTech	100-64
Human Flt-3 ligand	PeproTech	300-19
Human G-CSF	PeproTech	300-23
Human IGF1	PeproTech	100-11
Human IL-6	PeproTech	200-06
Human SCF	PeproTech	300-07
Human TPO	PeproTech	AF-300-18
LSRFortessa Cell Analyzer	BD Biosciences	NA
May-Grunwald solution	Generon	26250-01
Pannoramic 250 High Throughput Scanner	3DHISTECH	NA
Penicillin/Streptomycin	Thermo Scientific	15140-122
Shandon Cytospin 3	Thermo Scientific	NA
Stem Cell Medium medium	Stem Cell Technologies	9655