Elsevier

Cytotherapy

Volume 8, Issue 5, 2006, Pages 480-487
Cytotherapy

Differentiation of umbilical cord blood-derived multilineage progenitor cells into respiratory epithelial cells

https://doi.org/10.1080/14653240600941549Get rights and content

Background

Umbilical cord blood (UCB) has been examined for the presence of stem cells capable of differentiating into cell types of all three embryonic layers (i.e. endo-, ecto- and mesoderm). The few groups reporting success have typically confirmed endodermal potential using hepatic differentiation. We report differentiation of human UCB-derived multipotent stem cells, termed multilineage progenitor cells (MLPC), into respiratory epithelial cells (i.e. type II alveolar cells).

Methods

Using a cell separation medium (PrepaCyte-MLPC; BioE Inc.) and plastic adherence, MLPC were isolated from four of 16 UCB units (American Red Cross) and expanded. Cultures were grown to 80% confluence in mesenchymal stromal cell growth medium (MSCGM; Cambrex BioScience) prior to addition of small airway growth medium (SAGM; Cambrex BioScience), an airway maintenance medium. Following a 3–8-day culture, cells were characterized by light microscopy, transmission electron microscopy, immunofluorescence and reverse transcriptase (RT)-PCR.

Results

MLPC were successfully differentiated into type II alveolar cells (four of four mixed lines; two of two clonal lines). Differentiated cells were characterized by epithelioid morphology with lamellar bodies. Both immunofluorescence and RT-PCR confirmed the presence of surfactant protein C, a protein highly specific for type II cells.

Discussion

MLPC were isolated, expanded and then differentiated into respiratory epithelial cells using an off-the-shelf medium designed for maintenance of fully differentiated respiratory epithelial cells. To the best of our knowledge, this is the first time human non-embryonic multipotent stem cells have been differentiated into type II alveolar cells. Further studies to evaluate the possibilities for both research and therapeutic applications are necessary.

Introduction

Conflicting data in the literature initially led to debate over the presence of mesenchymal stromal cells (MSC) in umbilical cord blood (UCB) [1., 2., 3.]. UCB has since become an accepted source of MSC, stem cells capable of differentiating into cells of connective tissue lineage [4]. More recently, UCB has been examined for the presence of cells capable of differentiating into cell types of all three embryonic layers (i.e. endo-, ecto- and mesoderm). The few groups reporting success with UCB have used hepatic differentiation for confirmation of endodermal potential [5, 6].

Investigators working with stem cells derived from sources other than UCB have been successful with differentiation to an alternative cell type of endodermal origin, respiratory epithelium [7, 8]. Ali et al. [7] first demonstrated derivation of type II alveolar cells from murine embryonic stem cells (ESC) using an off-the-shelf medium designed for maintenance of fully differentiated respiratory epithelial cells. Panoskaltsis-Mortari et al. [8] later used similar techniques to differentiate murine BM-derived multipotent adult progenitor cells (MAPC) into ‘type II pneumocyte-like cells’. Following similar methods based upon their institutional experience with murine ESC [7], Samadikuchaksaraei et al. [9] recently were able to derive type II cells from human ESC.

Human multipotent stem cells from sources other than the embryo, however, have yet to be shown to have the in vitro capacity to yield respiratory epithelial cells. We report for the first time differentiation of human UCB-derived multipotent stem cells, termed multilineage progenitor cells (MLPC; BioE Inc., Saint Paul, MN, USA), into respiratory epithelial cells (i.e. type II alveolar cells). These efforts demonstrate further endodermal potential of human UCB-derived stem cells, and offer potential for research and clinical applications.

Section snippets

Cell culture

PrepaCyte-MLPC (BioE Inc.), an Ab-based cell separation medium, was added to a UCB unit (American Red Cross Cord Blood Program, Saint Paul, MN, USA). Following homo- and heterophilic aggregation of undesired cell populations and subsequent sedimentation to gravity, the supernatant containing stem cells was expressed. After overnight incubation (5% CO2/37°C) in a T-flask in MSC growth medium (MSCGM; Cambrex BioScience, Walkersville, MD, USA), non-adherent cells were washed, leaving adherent

Results

MLPC were isolated from four of 16 (25%) UCB units and then expanded. Expanded cells (CD9+ CD13+ CD29+ CD44+ CD73+ CD90+ CD105+ CD34 CD45 CD106) were successfully differentiated into cell types representative of each embryonic layer (e.g. osteocytes, hepatocytes, neuronal cells; data not shown).

Discussion

Investigators have previously reported success in deriving distal airway epithelium from murine ESC [7], murine marrow stem cells [8] and, more recently, human ESC [9]. Using culture methods similar to these investigators [7., 8., 9.], we were able to demonstrate successful differentiation of UCB-derived stem cells (MLPC) to type II alveolar cells. To the best of our knowledge this is the first time that human non-embryonic multipotent stem cells have been differentiated into type II alveolar

Acknowledgements

The authors thank Gib Ahlstrand of BioDale Imaging Center, University of Minnesota, Saint Paul, MN, for his assistance with the transmission electron microscopy.

References (20)

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