Berger M J, Adams S D, Tigges B M, Sprague S L, Wang X-J, Collins D P, McKenna D H
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
Cytotherapy. 2006;8(5):480-7. doi: 10.1080/14653240600941549.
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).
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.
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.
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.
人们已对脐带血（UCB）进行检测，以确定其中是否存在能够分化为所有三个胚胎层（即内胚层、外胚层和中胚层）细胞类型的干细胞。少数报告成功的研究小组通常通过肝细胞分化来确认内胚层潜能。我们报告了人脐带血来源的多能干细胞（称为多谱系祖细胞，MLPC）向呼吸道上皮细胞（即II型肺泡细胞）的分化情况。
使用细胞分离培养基（PrepaCyte-MLPC；BioE公司）和塑料贴壁法，从16个脐带血样本（美国红十字会提供）中的4个分离出MLPC并进行扩增。在添加小气道生长培养基（SAGM；Cambrex BioScience公司）（一种气道维持培养基）之前，将细胞培养在间充质基质细胞生长培养基（MSCGM；Cambrex BioScience公司）中，直至达到80%汇合度。经过3 - 8天的培养后，通过光学显微镜、透射电子显微镜、免疫荧光和逆转录酶（RT）-PCR对细胞进行鉴定。
MLPC成功分化为II型肺泡细胞（4个混合细胞系中的4个；2个克隆细胞系中的2个）。分化后的细胞具有含板层小体的上皮样形态。免疫荧光和RT-PCR均证实存在表面活性蛋白C，这是一种对II型细胞高度特异的蛋白质。
MLPC被分离、扩增，然后使用一种用于维持完全分化的呼吸道上皮细胞的现成培养基分化为呼吸道上皮细胞。据我们所知，这是人类非胚胎多能干细胞首次分化为II型肺泡细胞。有必要进行进一步研究以评估其在研究和治疗应用方面的可能性。
