Lu X, Alshemali S, de Wynter E A, Dickinson A M
Haematological Sciences, School of Clinical & Laboratory Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
Transfus Med. 2010 Jun;20(3):178-84. doi: 10.1111/j.1365-3148.2009.00981.x. Epub 2009 Nov 23.
Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34(-) UCB cells. CD34(+) cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34(-) cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic-fibroblast growth factor. Isolated CD34(+) cells were also cultured in the same medium. Adherent fibroblast-like cells at passage 3-4 were analyzed by fluorescence-activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4-5 weeks in culture, the cells from the CD34(-) fraction became confluent with flat and fibroblast-like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation-activated receptor gamma-2 (PPARgamma2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox-9 genes. In contrast, cells from the CD34(+) fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34(-) fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.
众所周知，脐带血（UCB）是干细胞的丰富来源，尤其是造血干细胞（HSCs）。最近，也已证明间充质干细胞（MSCs）存在于脐带血中。尽管在扩增后通过一部分表面抗原描述了间充质干细胞，但对于未分化的间充质干细胞的细胞表面表型知之甚少。因此，本研究的目的是阐明未分化的间充质干细胞是否存在于CD34（-）脐带血细胞中。通过磁性分选从脐带血单个核细胞（MNCs）中分离出CD34（+）细胞，并将CD34（-）细胞部分在含有10％胎牛血清（FCS）和碱性成纤维细胞生长因子的杜氏改良 Eagle培养基（DMEM）中培养。分离出的CD34（+）细胞也在相同培养基中培养。通过荧光激活细胞分选（FACS）分析传代3-4时贴壁的成纤维细胞样细胞的间充质干细胞标志物表达，并使用标准的成脂、成骨和软骨生成测定法研究其分化潜能。培养4-5周后，来自CD34（-）部分的细胞汇合，具有扁平和成纤维细胞样形态。这些细胞对间充质细胞标志物CD29、CD73和CD105呈阳性染色。在成脂分化中，细胞油红O染色呈阳性，并表达与脂肪生成相关的FABP4、脂肪酶和增殖激活受体γ-2（PPARγ2基因）。在成骨分化中，检测到钙积累和骨钙素。在软骨生成条件下生长的细胞对人聚集蛋白聚糖呈阳性染色，并表达II型胶原蛋白和Sox-9基因。相反，在相同培养条件下，来自CD34（+）部分的细胞未能产生任何具有间充质干细胞形态的细胞。我们的结果表明，脐带血含有仅存在于CD34（-）部分的间充质干细胞。这些间充质干细胞可以被诱导分化为至少三种谱系细胞类型，即脂肪细胞、成骨细胞和软骨细胞。
