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Cell Biology International (2008) 32, 815 (Printed in Great Britain)
Human mesenchymal stem cells isolated from the umbilical cord
Chun Qiao, Wenrong Xu*, Wei Zhu, Jiabo Hu, Hui Qian, Qing Yin, Runqiu Jiang, Yongmin Yan, Fei Mao, Huan Yang, Xingzhong Wang and Yongchang Chen
School of Medical Technology, Centre for Clinical Laboratory Medicine of Affiliated Hospital, Jiangsu University, Zhenjiang Key Institute of Clinical Laboratory Medicine, 301 Xuefu Road, Zhenjiang, Jiangsu 212013, P.R. China
Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2
Keywords: Mesenchymal stem cells, Umbilical cord, Differentiation.
*Corresponding author. Fax: +86 511 503 8449.
In recent years, multi-potential mesenchymal stem cells (MSCs) have become an attractive therapeutic tool because of their unique characteristics, such as their ability to be easily isolated and cultured and their high expansive potential ex vivo. Mesenchymal stem cells are able to be isolated from a wide variety of tissues, including bone marrow, adipose tissue, synovium, skeletal muscle, liver, cord blood, placenta and peripheral blood (Deans and Moseley, 2000; Jiang et al., 2002). However, bone marrow represents the main source of MSCs. Further more, MSCs have been shown to contain cells with multi-lineage potential under controlled in vitro conditions that mimic in vivo. These cells can differentiate into distinct types of mesenchymal cells including osteoblasts, chondroblasts, adipocytes and myoblasts, which contribute to the formation of mesenchymal tissues (bone, cartilage, muscle, marrow stroma, ligament, tendon, fat, dermis and connective tissue) (Schwartz et al., 2002; Xu et al., 2004). Therefore, further preclinical and clinical studies on the potential of mesenchymal stem cells are necessary for regenerative medicine, cellular immunotherapy and gene therapy (Wulf et al., 2006).
MSCs are not only present in bone marrow but also in the foetal environment (e.g., cord blood and placenta). Umbilical cord blood (UCB) is a source of additional stem cells for experimental and potentially clinical purposes. However, the presence of MSCs in UCB is controversial (Bieback et al., 2004; Kern et al., 2006). Placenta-derived cells also display multi-lineage differentiation potential similar to that of MSCs from bone marrow (Fukuchi et al., 2004; In 't Anker et al., 2004; Portmann-Lanz et al., 2006). The umbilical cord vein could be regarded as an alternative source of MSCs for experimental and clinical needs (Panepucci et al., 2004; Lu et al., 2006). Since MSCs are rarely found in bone marrow or foetal tissues, isolation and expansion of human mesenchymal stem cells appears to be crucial for their clinical applications. Our research attempts to establish a new method for isolating human MSCs derived from human umbilical cord, which could be able to differentiate into other tissues, such as osteogenic and adipogenic tissues, and to identify the characteristics of the isolated cells.
2 Materials and methods
2.1 Materials 2.1.1 Reagents Low-glucose Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and horse serum (HS) were purchased from Gibco (USA). Tyrode's solution and trypsin–EDTA were purchased from Sigma (St. Louis, MO). FITC-conjugated mouse anti-human antibodies (CD34, CD71, HLA-DR), PE-conjugated mouse anti-human antibodies (CD29, CD38, CD105, CD44 and HLA-I), FITC-conjugated mouse IgG1 and PE-conjugated mouse IgG1 were purchased from Becton Dickinson (San Jose, CA). Trizol reagent and reverse transcriptase–polymerase chain reaction (RT–PCR) kit were purchased from Invitrogen (Carlsbad, CA). Primers were synthesised by Shanghai Bio-Engineering Co. (Shanghai, China).
Low-glucose Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and horse serum (HS) were purchased from Gibco (USA). Tyrode's solution and trypsin–EDTA were purchased from Sigma (St. Louis, MO). FITC-conjugated mouse anti-human antibodies (CD34, CD71, HLA-DR), PE-conjugated mouse anti-human antibodies (CD29, CD38, CD105, CD44 and HLA-I), FITC-conjugated mouse IgG1 and PE-conjugated mouse IgG1 were purchased from Becton Dickinson (San Jose, CA). Trizol reagent and reverse transcriptase–polymerase chain reaction (RT–PCR) kit were purchased from Invitrogen (Carlsbad, CA). Primers were synthesised by Shanghai Bio-Engineering Co. (Shanghai, China).
2.2 Isolation of human umbilical cord MSCs
Fresh umbilical cords were collected from informed, consenting mothers and processed as quickly as possible. Moreover, the fresh umbilical cords were processed within the optimal processing period of 6
2.3 Morphology analysis
The cells harvested by trypsinisation were washed twice with PBS and then stained with the red fluorescent dye PKH26 (Sigma) according to the protocol of the supplier (Sigma). In brief, the washed cells were incubated with PKH26, which was added into 107 cells/ml at a concentration of 4
2.4 Flow cytometry
After the second passage, the cells were trypsinised (0.25% trypsin–EDTA), washed twice with 0.15
2.5 Growth curves
In order to compare the growth curves with the MSCs of human bone marrow, the cells isolated from umbilical cords were seeded in 24-well plates (0.5
2.6 Genetics and DNA contents
Colchicine was added to the suspension of umbilical cord cells during the logarithmic growth phase. After incubation of 4–6
2.7 Differentiation studies
The differentiation of the umbilical cord MSCs was assessed in cultures of the third passage. The cells were cultured in a medium which contained either osteogenic (0.1
2.8 Reverse transcriptase–PCR (RT–PCR)
Before and after differentiation, the total RNA of MSCs generated from both human bone marrow and the umbilical cord were extracted by Trizol reagent (Invitrogen, USA). The cDNA was synthesised by using 4
Specific primers for target and control gene
3.1 Morphology and surface antigens
After the initial 3
Morphological appearance of human umbilical cord MSCs. (A–C) The appearance and growth of MSCs colonies after 7, 10, and 15
PKH26 and DAPI staining of human umbilical cord MSCs. (A,B) Umbilical cord MSCs appeared red in fluorescence within 20
The surface antigens of human umbilical cord MSCs. (A) Umbilical cord MSCs were positive for CD29, CD44, CD95, CD105 and HLA-I. (B) Umbilical cord MSCs were negative for CD34, CD38, CD71 and HLA-DR. The results confirmed that the cells were a kind of MSCs but non-hematopoietic cells.
3.2 Growth characteristics
The time for 1 passage was about 4–6
Growth curve of umbilical cord MSCs and bone marrow MSCs. According to the growth curve, the population doubling time of umbilical cord MSCs was 26
3.3 Genetics and cell cycle
The karyotype of human umbilical cord MSCs was normal (Fig. 5). The DNA contents also revealed similar results. Analysis for the cell cycle indicated that the third phases were: G
Karyotype of umbilical cord MSCs. The karyotype of human umbilical cord MSCs was normal.
DNA contents of human umbilical cord MSCs. (A–D) DNA contents of human umbilical cord MSCs of the 3rd, 10th, 16th and 20th passage, respectively. The results of the 16th and 20th passage indicated that they had propagated longer than MSCs from the other adult tissues. The cells became senile and diverse after the 20th passage yet proliferated slowly and passaged serially.
3.4 Differentiation of MSCs into osteocytes and adipocytes
With osteogenic supplementation the differentiation was apparent after 1
Differentiation potential of umbilical cord MSCs. (A,B) Results of alkaline phosphatase and Von Kossa detection in cell cultures growing within 2
Gene expression of adult MSCs. Specific genes, such as nucleostemin, BMI-1, BMP-3 and PPARγ2 were detected from the cDNA of differentiated and undifferentiated umbilical cord MSCs. Adult MSCs associate genes, nucleostemin and BMI-1 were expressed in all MSCs. However, BMP-3, an osteogenic marker gene, was only expressed in the MSCs for osteogenic differentiation. PPARγ2, an adipogenic marker gene in the MSCs, was expressed for adipogenic differentiation. B-LCL and PBL were attached as negative controls for BMI-1 and nucleostemin, respectively. Endogenous “housekeeping” gene GAPDH was used as an internal control.
3.5 Expression of specific genes
Fig. 8 shows that adult MSCs associate genes nucleostemin and BMI-1 were expressed in all MSCs. However, BMP-3, an osteogenic marker gene, was only expressed in the MSCs for osteogenic differentiation. In addition, PPARγ2, an adipogenic marker gene in the MSCs, was tested for adipogenic differentiation. B lymphoblastoid cell line (B-LCL) and peripheral blood lymphocytes (PBL) were attached as negative controls for BMI-1 and nucleostemin, respectively (n
Mesenchymal stem cells offer a lot of promise for developing new alternative cell-based therapeutics. These unique cells possess two major features: their ability for self-renewal and differentiation potential. Furthermore, cells with mesenchymal stem characteristics can be derived and propagated in vitro from different organs and tissues (brain, heart, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas) (Beltrami et al., 2003; Alison et al., 2004; Laugwitz et al., 2005; Meirelles et al., 2006). Until now, it has been difficult to isolate human MSCs from most of the healthy tissues of the above organs. MSCs are generally isolated from an aspirate of bone marrow harvested from the superior iliac crest of the pelvis, the tibia and femur, and the thoracic and lumbar spine.
Their pharmacological importance is related to four points of mesenchymal stem cells and due to their ability to secrete biologically important molecules, express specific receptors, to be genetically manipulated and their susceptibility to molecules that modify their natural behaviour (Beyer Nardi and da Silva Meirelles, 2006). MSCs from the different tissue types present great potential in cellular therapies and in our quest for longevity (Majka et al., 2005). With the conventional limitation of MSCs isolation in mind, we have attempted to search for new sources of MSCs. We adopted the adherent culture method of human umbilical cords of term birth and have successfully isolated and identified a population of mesenchymal stem cells from the human umbilical cord. Moreover, we found the fresher the umbilical cord, the faster the rate of cell proliferation. The experiments were performed four times with consistent results. MSCs are non-hematopoietic stem cells with pluripotency. Umbilical cord is based upon body stalk and enwrapped by amnion, which consists of yolk bag, allantoid, two pieces of cord arteries and one piece of cord vein. The amnion contains ectoderm and mesoblast from the embryo. Usually, endothelial cells which are present in primary cultures do not affect the final outcome as the cells can not adhere to a plastic surface and proliferate for a long time without essential growth factors. Our results suggest that umbilical cord MSCs may originate from the sub-endothelial layer of the umbilical cord vein (Romanov et al., 2003). Whether the MSCs originate from amnion or smooth muscle requires further investigation. FCM analysis showed that these cells expressed the same surface antigens as bone marrow MSCs. The positive ratio weakened when the cells passaged for several months (not shown). The results confirmed that these cells were a kind of MSCs, but non-hematopoietic cells.
Martin et al. (2002) reported that MSCs were able to proliferate in vitro. The umbilical cord stem cells tendency for colony growth with long passages (4
To investigate the differentiation potential of MSCs derived from the umbilical cord, we used MSCs of the third passage to culture in the conditions that favoured osteogenic and adipogenic differentiation of MSCs. Results indicated that the MSCs could differentiate into osteocytes and adipocytes that present ALP, Von Kossa, Oil-Red-O positive staining in plasma and expressed, in addition, different osteogenic and adipogenic marker genes such as BMP-3 and PPARγ2. Our experiments revealed that the MSCs cultured in adipogenic differentiation medium led to the appearance of rounded cells. These cells presented numerous fat vacuoles in cytoplasm visualised by Oil-Red-O staining.
Some associated genes were selected to search for the differences between MSCs derived from bone marrow and those derived from the umbilical cord. Nucleostemin is a novel p53 binding protein localised in the nucleoli of stem cells but absent from committed and terminally differentiated cells (Tsai and Mckay, 2002). BMI-1 is capable of self-renewal and could restrain the senescence of MSCs (Bea et al., 2001). The nucleostemin and BMI-1 mRNA was expressed in the human umbilical cord MSCs as well as bone marrow MSCs. In order to confirm the specificity of RT–PCR products for nucleostemin and BMI-1, DNA sequencing of PCR products was performed. The results indicated that products of RT–PCR for nucleostemin and BMI-1 were completely homologous to the sequence from GenBank (NM175580, NM005180). These findings suggested that MSCs of different sources had similar characteristics of gene expression.
In conclusion, umbilical cord MSCs have been established and characterised as a new human MSC cell line. This is a significantly different approach from previous reports in which the vasculature and its surrounding tissue have been discarded. We have taken a method for isolating MSCs without collagenase which may affect the proliferation and differentiation of MSC. Our results indicate that the isolation method is significantly different from others reported (Wang et al., 2004; Baksh et al., 2007). However, the identification of umbilical cord MSCs is similar to theirs in differentiation potential. These cells retain the morphologic, biologic and cytogenetic characteristics of human bone marrow mesenchymal stem cells in vitro, with expression of nucleostemin and BMI-1. The umbilical cord MSCs may provide evidence for the theory that stem cells might originate from many tissues. Compared to bone marrow MSCs, umbilical cord MSCs are easier to isolate and expand. Our harvesting procedure is more consistent and yields a greater number of relevant cells than results achieved from the other more primitive tissues. Our findings indicate that umbilical cord MSCs are a novel source of adult mesenchymal stem cells. Umbilical cord MSCs may play an important role in applications and experimental research of adult human MSCs.
We thank Professor Qi Ming and Olivia Lindsey for helpful discussion and critical reading of the manuscript. This work was supported by National Natural Science Foundation of China grant 30471938, Natural Science Foundation of Ministry of Public Health of China Grant WKJ 2005-2-024, Jiangsu Province's Outstanding Medical Academic Leader program, Foundation of Zhenjiang Key Institute of Clinical Laboratory Medicine Grants SH2006066, SH2006070, The Natural Science Foundation of the Jiangsu Province, grant no. BK2007705, and BK2007092.
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Received 2 February 2007/19 June 2007; accepted 10 August 2007doi:10.1016/j.cellbi.2007.08.002