Abstract
Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.
Highlights
Red blood cells (RBCs) make up 40 to 50 percent of the average human blood volume and are the most commonly transfused blood product, with 40,000 RBC units (,220 mL) used in the United States every day [1]
The mean RBC number concentration is 56109/mL of whole blood [17], which is much higher than that expected in hematopoietic stem cells (HSCs)-derived RBC cultures; it was assumed that this method would deoxygenate the cultures well or better
Given the goal of producing RBCs for safe transfusions into humans, is it desirable for the final product to contain functioning RBCs and that it is devoid of contaminating cells
Summary
Red blood cells (RBCs) make up 40 to 50 percent of the average human blood volume and are the most commonly transfused blood product, with 40,000 RBC units (,220 mL) used in the United States every day [1]. A number of recent studies have suggested the possibility of ex vivo erythrogenesis from hematopoietic stem cells (HSCs) isolated from peripheral blood, bone marrow, and umbilical cord collected following delivery [2,3]. This approach could provide the basis for large-scale RBC production, in combination with a suitable protocol for HSC expansion and staged erythrocytic differentiation. Obtaining mature and functional erythrocytes from cultured HSC populations with high purity remains a challenge
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