Isolation and Characterization of Umbilical Cord Blood-Derived Multipotent Stem Cells Arising from Adherent CD45+/CD34+ Cell Subset.

Introduction: Using a cell separation medium (PrepaCyte-MLPC, BioE, Inc.) and plastic adherence, umbilical cord blood (UCB)-derived multipotent stem cells were isolated, expanded, and characterized. These stem cells, termed multi-lineage progenitor cells (MLPCs), are the only such cell type from any source demonstrated to be CD45+/CD34+ upon initial isolation.

Methods: PrepaCyte-MLPC, an antibody-based cell separation medium, is added to an UCB unit (American Red Cross Cord Blood Program). Following homo- and heterophilic aggregation of undesired cell populations and subsequent sedimentation to gravity, the supernatant containing stem cells is expressed. After overnight incubation in a T-flask in MSCGM (Cambrex, Inc.), non-adherent cells are washed, leaving adherent cells to expand in culture. As MLPC colonies are observed, cells are further enriched by detachment (PBS/0.1% EGTA) and transfer to a new T-flask.

Results: MLPCs were isolated and expanded from 3 of 10 UCB units. Cells were initially positive for CD45, CD34, CD133, SSEA-3, SSEA-4, and several MSC markers. Several days into culture, expression of HSC/ESC markers was lost. MSC marker expression was maintained; cells remain positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105. These MLPC lines have been cultured through at least 16 passages with no apparent impact on expansion or differentiation potential. Cell lines have been successfully differentiated into cell types representative of each embryonic layer (i.e., adipocytes, osteocytes, myocytes, hepatocytes, and endothelial and neural cells). Additional experiments and limited-dilution cloning are underway, and further studies (cytogentic/molecular and animal teratoma studies) are planned.

Conclusions: UCB-derived MLPCs were isolated as a rare population of adherent, initially CD45+/CD34+ cells with lymphoid morphology. Following successful expansion in culture, multi-potency was demonstrated. MLPCs may serve a role in bone marrow transplantation (for malignant and non-malignant disease) and regenerative medicine; clinical studies are in an early phase of development.