A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo
January 10th, 2025
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.
Our research focuses on leukemia, specifically myelodysplastic syndromes or myelodysplastic neoplasia. It's a stem cell disease originating from hematopoietic stem cells. It affects various blood cell lineages, particularly the erythroid lineage, which is responsible for heart blood cell production.
Disruption of this lineage can lead to the anemia, which is commonly seen in MDS patients. The pipeline we provide in this article allows a robust and simplified method to assess human erythroblast efficiency in vitro upon gene modification or drug treatment. Hematopoietic stem cells are present from birth to the last moment of our lives.
There is no more proliferative tissue in our organism with trillions of cells produced every day. There are still so many things to understand, so we'll keep studying these amazing cells in healthy amino tissues with a strong emphasis on proteostasis and RNA biology. To begin, thaw 100, 000 CD34+UCB cells by dipping the vial for 30 seconds in a water bath set to 37 degrees Celsius.
Add one milliliter of defrosting medium dropwise to the vial, then gently resuspend the cells by pipetting up and down. Transfer the resuspended cell suspension drop by drop into five milliliters of defrosting medium. Then centrifuge the cell suspension for eight minutes at 320 G at room temperature.
Wash the cells in 10 milliliters of fluorescence-activated cell sorting or FACS buffer with DNAse. After centrifuging the cells again, resuspend the pellet in two milliliters of stimulation media. Transfer two milliliters of cell suspension into a 24-well plate and incubate the plate for four to six hours at 37 degrees Celsius.
Then collect the cells in fluorescence-activated cell sorting tubes and top them up with one milliliter of stem cell medium. After thawing the virus, add the required volume to stimulation media to achieve a multiplicity of infection of 30. Resuspend the pelleted cells in virus-containing media at a density of 100, 000 cells per 100 microliters of virus and transfer them into one well of a 96-well plate.
Add PBS to the empty wells and incubate overnight at 37 degrees Celsius. Then transfer the full volume of cell suspension from the well of the 96-well plate into FACS tubes with one milliliter of stem cell medium. Wash the well and the pipette tip with stem cell medium to add two milliliters of stem cell medium to the FACS tube.
After centrifuging the FACS tube, discard the supernatant and resuspend the cell pellet in two milliliters of expansion media. Transfer the suspension into a well of a 24 well plate and incubate the cells. To begin, transfer the full volume of CD34+cells transduced with the EGFP reporter to a separate FACS tube.
Centrifuge at 320 G for eight minutes at room temperature. Resuspend the pelleted cells in 100 microliters of FACS buffer. Add five microliters of CD34 antibody per tube and incubate the tubes in the dark at four degrees Celsius for 20 minutes.
Then resuspend the cells in one milliliter of FACS buffer containing DAPI and centrifuge for eight minutes at 320 G at room temperature. After resuspending the cells in 200 microliters of FACS buffer containing DAPI, filter the cell suspension through a 40 micrometer strainer prior to sorting. Next, add 0.5 microliters of CD34 antibody to one drop of beads in 100 microliters of PBS.
Incubate the mixture in the dark at four degrees Celsius for 20 minutes. Then add one milliliter of PBS to the mixture. Centrifuge for eight minutes at 320 G at room temperature.
Discard the supernatant and resuspend the pellet in 500 microliters of PBS. Next, add GFP-negative and GFP-positive leukemic cells to a FACS tube. Wash the cells in one milliliter of FACS buffer.
After centrifuging the cells, resuspend 100, 000 GFP-negative cells and 100, 000 GFP-positive cells, each in 500 microliters of FACS buffer. Filter the cell suspension through a 40 micrometer strainer prior to sorting. Then collect the sorted cells in stem cell medium.
Centrifuge the cells for eight minutes at 320 G at room temperature. Then resuspend the cells in three milliliters of erythroid differentiation media. Now dispense one milliliter of the resuspended cells into three wells of a 24-well plate.
Fill all the outer wells with PBS. To refresh the media, ensure the cells have settled at the bottom of the plate. Gently lean the plate and remove 500 microliters of media.
Then add 500 microliters of 2X cytokine erythroid differentiation media to refresh the plate. On day six, transfer the cells to a 12-well plate and add one milliliter of media to each well to reach a final volume of two milliliters. Collect 200 microliters of cells from each well of the 12-well plate.
Replace the removed volume with 250 microliters of 2X erythroid differentiation media. Add one milliliter of FACS buffer to each collection tube. After centrifuging the tubes, resuspend the pelleted cells in 100 microliters of FACS buffer.
Add one microliter each of CD71, CD34, and CD235a antibodies to the tubes. Then resuspend the cells in one milliliter of FACS buffer containing DAPI. Centrifuge the tubes for eight minutes at 320 G at room temperature.
Resuspend the cells in 200 microliters of FACS buffer with DAPI. Set DAPI versus side scatter or SSC to exclude dead cells. Set SSC height versus SSC area to exclude aggregates and select single cells.
Set forward scatter area versus SSCA to remove debris based on size. Set CD235a versus CD71. Set CD34 versus SSCA.
Run beads GFP-positive cells and DAPI-stained cells. Compensate the readings and apply the compensation to the cytometer settings. CD34-positive cells from umbilical cord blood exhibited an increase in Annexin V positive apoptotic cells from 14.4%on day four to 34.2%on day 17.
The bar graph shows a significant increase in the percentage of Annexin V-positive cells by day 17, reaching approximately 40%
View the full transcript and gain access to thousands of scientific videos
Overview
This study investigates the differentiation of CD34+ hematopoietic stem and progenitor cells ex vivo, addressing the critical processes involved in erythropoiesis and its implications for treating related disorders like myelodysplastic syndromes. By providing a simplified method to evaluate erythroblast efficiency, the research aims to enhance understanding of the disturbances leading to anemia in these patients.
Key Study Components
Research Area
- Erythropoiesis
- Myelodysplastic syndromes
- Hematopoietic stem cell biology
Background
- Myelodysplastic syndromes are stem cell diseases affecting blood cell lineages.
- Disruption of the erythroid lineage can lead to anemia.
- Understanding hematopoietic stem cells is crucial as they persist throughout human life.
Methods Used
- Thawing and resuspending CD34+ UCB cells
- Cell transduction with gene modifications
- Fluorescence-activated cell sorting (FACS)
Main Results
- CD34+ cells exhibited increased apoptotic rates over time.
- Efficient differentiation into erythroblasts was demonstrated.
- The specific methods developed allow for assessment post gene modification or drug treatment.
Conclusions
- The study demonstrates a robust method for differentiating hematopoietic cells that may lead to more effective treatments for anemia caused by myelodysplastic syndromes.
- This work underscores the importance of understanding cell differentiation in stem cell biology.
Frequently Asked Questions
Executive Industry Relevance
Efficient assessment of human erythropoiesis is critical for de-risking early-stage hematology drug discovery and understanding mechanisms underlying anemia and myelodysplastic syndromes. This pipeline enables robust, quantitative evaluation of erythroid differentiation and gene function in both healthy and disease-relevant human systems. The approach supports predictive confidence in target validation and informs portfolio decisions for therapies modulating erythropoiesis.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of gene function and pathway impact on erythroid lineage commitment.
- Supports biological de-risking by quantifying effects of genetic or compound perturbations on erythropoiesis.
- Facilitates functional target validation in primary human hematopoietic stem and progenitor cells.
- Provides mechanistic insight into disease-relevant disruptions such as those seen in myelodysplastic syndromes.
Screening & Assay Development
- Establishes validated in vitro and ex vivo systems for compound screening affecting erythroid differentiation.
- Enables reproducible, quantitative readouts using flow cytometry and marker-based cell identification.
- Supports assay standardization for cross-study and cross-team comparability.
- Prepares scalable platforms for evaluating gene modulation and drug rescue strategies.
Translational & Preclinical Research
- Aligns in vitro findings with ex vivo patient-derived models for translational continuity.
- Enables risk-adjusted advancement of candidates with demonstrated effects on erythropoiesis.
- Supports biomarker development by tracking differentiation and apoptosis markers across timepoints.
- Provides mechanistic de-risking for preclinical candidate selection in hematology portfolios.
Pipeline & Workflow Integration
This method integrates from early discovery through lead identification and preclinical validation in hematology-focused pipelines.
- Discovery Biology: Quantifies gene and compound effects on erythroid lineage specification and maturation.
- Screening: Delivers standardized, reproducible flow cytometry-based assays for compound and genetic screening.
- Analytics: Provides quantitative outputs such as apoptosis rates and marker expression for comparative analysis.
- Translational Research: Bridges in vitro and ex vivo findings to patient-relevant disease models.
- Enterprise Reuse: Offers a modular, reusable workflow adaptable to diverse gene targets and compound libraries.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation and mechanistic understanding of erythropoiesis.
- Operational Value: Standardizes workflows for reproducibility and scalability across R&D teams.
- Strategic Value: Enables informed go/no-go decisions and reduces late-stage biological risk in hematology programs.
- Portfolio Impact: Supports risk-adjusted prioritization of targets and compounds with validated erythroid effects.
Implementation Considerations
- Requires expertise in hematopoietic stem cell culture and flow cytometry analysis.
- Needs access to FACS instrumentation and validated antibody panels for erythroid markers.
- Demands cross-team standardization of gating strategies and data interpretation.
- Adaptable to both healthy donor and patient-derived samples for broader applicability.
- Dependent on robust cell sourcing and quality control for reproducible outputs.
