Isolation of three important types of stem cells from the same samples of banked umbilical cord blood

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine, numerous umbilical cord blood banks have been established. In this study, we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs, MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs), slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48?h supernatant transferring, we successfully isolated MSCs which expressed CD13, CD44 and CD90 while CD34, CD45 and CD133 negative, had typical fibroblast-like shape, and was able to differentiate into adipocytes; EPCs which were CD34, and CD90 positive, CD13, CD44, CD45 and CD133 negative, adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium.     

Mesenchymal stem cells can be obtained from the human saphena vein

Mesenchymal stem cells (MSC) can be isolated from many sites adults and the fetus. Cells with osteoblastic, chondrogenic, leiomiogenic and stromogenic potentials have been obtained from the bovine artery wall, and we now show that MSC can be isolated also from the adult human vein wall. Cells detached from internal surface of the saphenous vein are cultured in vitro for 2-3 weeks and replated weekly. The culture forms a semi-confluent layer of spindle-shaped cells that are CD13(+), CD29(+), CD44(+), CD34(-), CD45(-), CD14(-), CD133(-), CD31(-), CD33(-), CD54(+), CD106(-), CD90(+), KDR(-), cadherin-5-, HLA class I(+) and HLA-DR- and differentiate in vitro into osteoblasts, chondrocytes and adipocytes. Gene expression, when compared with seven other normal tissues, shows strong similarity with MSC obtained from other sources. Three genes more expressed in saphenous MSC than in the other two MSC are related to angiogenesis, and the expression of two of them is shared by endothelial cells. These results demonstrate that the human vein wall contains mesenchymal cells with morphologic features, immunophenotypic markers, gene expression profile and differentiation potential that are similar to MSC obtained from the bone marrow and from the umbilical vein.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.     

Human mesenchymal stem cells isolated from the umbilical cord

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 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

脐静脉和骨髓来源的间充质干细胞的比较研究

间充质干细胞(MSCs)的来源有限,成人骨髓是MSCs的主要来源,这极大地限制了其在实验和临床中的应用。为拓宽MSCs来源,从细胞形态、生长特性、免疫表型和多向分化能力等四个方面对人脐静脉来源和成人骨髓来源的间充质干细胞进行了比较研究。结果表明,人脐静脉来源和成人骨髓来源的 MSCs具有相似的生物学特征,成纤维细胞样形态生长,并具有强大的体外扩增和多向分化能力。人脐静脉来源的MSCs可替代成人骨髓MSCs,作为满足实验和临床需要的重要来源。     

间充质干细胞对免疫细胞的抑制作用及其机制

间充质干细胞是一群来源于发育早期中胚层的具有自我更新和多向分化潜能的干细胞,具有分化为脂肪细胞、肝细胞、成骨细胞、软骨细胞、神经细胞等多种细胞的能力.近年来的相关研究表明,间充质干细胞具有低免疫原性,它可以通过抑制淋巴细胞的增殖、抑制抗原呈递细胞分化成熟及功能发挥、抑制细胞毒性T淋巴细胞的形成、增加调节性T细胞比例等多种途径发挥免疫调节作用,从而成为移植领域、各种退行性和衰竭性疑难病症的替代治疗的研究热点.本文就间充质干细胞对免疫细胞的抑制作用及其机制的研究进展进行综述.     

A simple and biosafe method for isolation of human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVEC_S) are used as an irreplaceable tool for the study of vascular diseases. However, the technicians who isolate HUVECs are largely exposed to potential infectious threats. Here we report the development of a specialized instrument to protect researchers from known or unknown infectious agents when they operate on human umbilical cords. This instrument can be assembled by common laboratory supplies and adapted to accommodate umbilical cords of different lengths. When the cord is enclosed within the instrument, the risk of sample contamination and operator infection is greatly reduced. Using our instrument, endothelial cells were successfully isolated from human umbilical veins without contamination. The cells were verified by their cobblestone-like morphology and by immunofluorescence staining (Factor VIII and CD31 positivity and α-SMA negativity). Our instrument simplifies and optimizes the cell extraction process, and most importantly elevates the biosafety to a higher level during the isolation of human umbilical vein endothelial cells.     

Isolation and characterization of mesenchymal stem cells from caprine umbilical cord tissue matrix

Cord tissue fills the umbilical cord around the blood vessels and contains types of stem cells (mesenchymal stem cells or MSCs) that are not generally found in cord blood. MSCs are the stem cells that give rise to many of the “support tissues” in the body, including bone, cartilage, fat and muscle. Umbilical Cord Tissue cells (UCTs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes have been previously isolated from different species including human, canine, murine, avian species etc. The present study documents the existence of similar multipotential stem cells in caprine UCTs having similar growth and morphological characteristics. The cells were isolated from caprine umbilical cord and cultivated in DMEM (low glucose) supplemented with 15% FBS, L-glutamine and antibiotics. Primary culture achieved confluence in 5–7 days having spindle shaped morphology. The cells were morphologically homogeneous, showed robust proliferation ability with a population doubled time of 92.07 h as well as normal karyotype. In vitro self-renewal capacity was demonstrated by colony-forming unit assay (CFU). The cells expressed MSC specific markers and showed multi-differentiation capability into adipogenic and osteogeneic. The results indicated that caprine UCTs (cUCTs) were isolated and characterized from umbilical cord tissue which can be used for tissue regeneration.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

In vitro differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells

In addition to long-term self-renewal capability, human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Of particular concern is hepatogenic potential that can be used for liver-directed stem cell therapy and transplantation. In this study, we have investigated whether human umbilical cord blood (UCB)-derived MSCs are also able to differentiate into hepatocyte-like cells. MSCs isolated from UCB were cultured under the pro-hepatogenic condition similar to that for bone marrow (BM)-derived MSCs. Expression of a variety of hepatic lineage markers was analyzed by flow cytometry, RT-PCR, Western blot, and immunofluorescence. The functionality of differentiated cells was assessed by their ability to incorporate DiI-acetylated low-density lipoprotein (DiI-Ac-LDL). As the cells were morphologically transformed into hepatocyte-like cells, they expressed Thy-1, c-Kit, and Flt-3 at the cell surface, as well as albumin, alpha-fetoprotein, and cytokeratin-18 and 19 in the interior. Moreover, about a half of the cells were found to acquire the capability to transport DiI-Ac-LDL. Based on these observations, and taking into account immense advantages of UCB over other stem cell sources, we conclude that UCB-derived MSCs retain hepatogenic potential suitable for cell therapy and transplantation against intractable liver diseases.     

Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth suplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65 ± 0.2% and CD106/VCAM 7.73±0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.     

A simple and serum-free protocol for cryopreservation of human umbilical cord as source of Wharton’s jelly mesenchymal stem cells

Mesenchymal stromal cells (MSCs) show promise in cell-based transplantations and regenerative medicine applications. MSCs from Wharton’s jelly (WJ) of umbilical cord can be easily harvested and exhibit greater proliferative activity than bone marrow MSCs. It is important to develop a practical cryopreservation technique to effectively store umbilical cord for potential future applications. Successful cryopreservation would allow access to umbilical cord from the same donor for repeated WJ MSC-based transplantations. For therapeutic applications, one should be able to obtain clinically-relevant quality and quantity of MSCs from cryopreserved tissues. In this study, we optimised a serum-free formulation of 10% dimethyl sulfoxide (DMSO) and 0.2 M sucrose for cryopreservation of umbilical cord tissue. Slow freezing and rapid thawing were adopted. MSCs harvested from WJ of cryopreserved umbilical cord could undergo robust expansion, differentiate to mesodermal lineages and express MSC-characteristic surface antigens. The cumulative cell yield, however, was less compared to corresponding fresh cord tissue.     

Mesenchymal stromal cells from unconventional model organisms

Mesenchymal stromal cells (MSCs) are multipotent, plastic, adherent cells able to differentiate into osteoblasts, chondroblasts and adipocytes. MSCs can be isolated from many different body compartments of adult and fetal individuals. The most commonly studied MSCs are isolated from humans, mice and rats. However, studies are also being conducted with the use of MSCs that originate from different model organisms, such as cats, dogs, guinea pigs, ducks, chickens, buffalo, cattle, sheep, goats, horses, rabbits and pigs. MSCs derived from unconventional model organisms all present classic fibroblast-like morphology, the expression of MSC-associated cell surface markers such as CD44, CD73, CD90 and CD105 and the absence of CD34 and CD45. Moreover, these MSCs have the ability to differentiate into osteoblasts, chondroblasts and adipocytes. The MSCs isolated from unconventional model organisms are being studied for their potential to heal different tissue defects and injuries and for the development of scaffold compositions that improve the proliferation and differentiation of MSCs for tissue engineering.     

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP-EPCs express CD133 but not CD14. Also, unlike CACs, HPP-EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind-limb model of ischemia in mice (BALB/C-nu) in vivo.     

间充质干细胞分化为内皮细胞的意义和细胞学基础

间充质干细胞(mesenchymalstemcells,MSCs)主要存在于骨髓中,是多潜能干细胞,在脐血、外周血、脂肪、皮肤等多种组织中也相继分离出MSCs。MSCs具有独特的免疫特性,在异种异体环境内长期存在,使其临床应用前景更为广泛。目前,MSCs的分离培养、诱导分化及鉴定体系已趋成熟,理论上可分化为所有中胚层来源的细胞,内皮细胞来源于中胚层,因此MSCs具有分化为内皮细胞的可能性。本文对MSCs内皮分化意义和细胞学基础及其新近的研究进展作一综述。     

Isolation,characterisation and comparative analysis of human umbilical cord vein perivascular cells and cord blood mesenchymal stem cells

Perivascular cells are known to be ancestors of mesenchymal stem cells (MSCs) and can be obtained from heart, skin, bone marrow, eye, placenta and umbilical cord (UC). However detailed characterization of perivascular cells around the human UC vein and comparative analysis of them with MSCs haven’t been done yet. In this study, our aim is to isolate perivascular cells from human UC vein and characterize them versus UC blood MSCs (UCB-MSCs). For this purpose, perivascular cells around the UC vein were isolated enzymatically and then purified with magnetic activated cell sorting (MACS) method using CD146 Microbead Kit respectively. MSCs were isolated from UCB by Ficoll density gradient solution. Perivascular cells and UCB-MSCs were characterized by osteogenic and adipogenic differentiation procedures, flow cytometric analysis [CD146, CD105, CD31, CD34, CD45 and alpha-smooth muscle actin (α-SMA)], and immunofluorescent staining (MAP1B and Tenascin C). Alizarin red and Oil red O staining results showed that perivascular cells and MSCs had osteogenic and adipogenic differentiation capacity. However, osteogenic differentiation capacity of perivascular cells were found to be less than UCB-MSCs. According to flow cytometric analysis, CD146 expression of perivascular cells were appeared to be 4.8-fold higher than UCB-MSCs. Expression of α-SMA, MAP1B and Tenascin-C from perivascular cells was determined by flow cytometry analysis and immunfluorescent staining. The results appear to support the fact that perivascular cells are the ancestors of MSCs in vascular area. They may be used as alternative cells to MSCs in the field of vascular tissue engineering.