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Prospective identification of erythroid elements in cultured peripheral blood
J L Miller1, J M Njoroge, A N Gubin
1Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. jm7f@nih.gov
Abstract:
We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48- cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48- cells increased to 34.7 + 14.9% (p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcellulose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.
Insights
Researchers identified erythroid cell generation from peripheral blood mononuclear cells (PBMC) by tracking CD48 protein expression. This flow cytometry method offers a reliable way to study human erythropoiesis in real-time.
Area of Science:
- Hematology
- Cell Biology
- Immunology
Background:
- Peripheral blood mononuclear cells (PBMC) are a source for studying hematopoiesis.
- Erythropoiesis, the development of red blood cells, is a complex process.
- Identifying specific cell populations during erythropoiesis is crucial for research.
Purpose of the Study:
- To develop a prospective method for identifying erythroid cell generation from cultured PBMC.
- To utilize cell surface protein expression for tracking erythroid differentiation.
- To establish a reliable flow cytometry approach for studying human erythropoiesis.
Main Methods:
- Culturing unpurified PBMC from normal volunteers in suspension.
- Supplementing cultures with or without erythropoietin (EPO).
- Monitoring CD48 cell surface protein expression via flow cytometry.
- Assessing CD34, CD71 expression, cell morphology, gamma-globin transcription, and colony formation.
Main Results:
- CD48 expression profiling enabled flow cytometric identification of EPO-responsive erythroid precursors.
- In EPO-supplemented cultures, CD48- cells increased significantly (mean 34.7%) after 14 days.
- Coordinated expression of CD34, CD71, and gamma-globin transcription confirmed erythroid maturation.
Conclusions:
- Monitoring CD48 expression is a simple and reliable method for prospective identification of erythroid cells.
- Flow cytometry of bulk cultured PBMC allows for real-time study of human erythropoiesis.
- This approach facilitates the investigation of erythroid cell development and differentiation.