In Vitro Cell.Dev.Biol.-Animal (2009) 45:23–27 DOI 10.1007/s11626-008-9155-4

REPORT

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord Sachin S. Kadam & Shubha Tiwari & Ramesh R. Bhonde

Received: 9 July 2008 / Accepted: 13 October 2008 / Published online: 5 December 2008 / Editor: J. Denry Sato # The Society for In Vitro Biology 2008

Abstract The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child’s birth; however, its importance as a “store house” of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton’s jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for vonWillibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

S. S. Kadam : S. Tiwari : R. R. Bhonde (*) National Center for Cell Science, Ganeshkhind, Pune 411007, India e-mail: [email protected]

Keywords Umbilical cord vein . Human umbilical cord vascular endothelial cells . Wharton’s jelly . Human umbilical cord matrix stem cells The umbilical cord is a connection between the growing fetus and the maternal blood circulation. The average length of the umbilical cord is about 50 cm, but it is subject to great variation (20 to 120 cm; William et al. 1995). It has an average diameter of 1–2 cm. The histology of umbilical cord reveals a vein and two arteries with average diameter of about 7–8 and 3–4 mm, respectively. These blood vessels are surrounded by mucoid connective tissue, called Wharton’s jelly. Endothelial cells (ECs) form a monolayer that lines the entire vascular system (endothelium). Their structural and functional integrity is fundamental to the maintenance of vessel wall homeostasis and the local and systemic circulation functions. Common research tools for the study of atherosclerosis are arterial and venous ECs of human umbilical cord since 1973 (Jaffe et. al. 1973; Ko et al. 1995). Umbilical cords are easily available, whereas human aortic tissue can only be obtained during surgical or invasive interventions. At the time of delivery, when the umbilical cord is cut and clamped, the arteries constrict much more than vein. Thus, it is possible to employ the umbilical cord vein as a source of vascular endothelial cells. Two types of stem cells have been found in umbilical cord: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). HSCs from umbilical cord blood (UCB) have already been proven to be useful in treating various hematological disorders (Laughlin et al. 2001; Hayani et al. 2007). However, the presence of MSCs in UCB is controversial. Some researchers succeeded in isolating these cells (Erices et al. 2000; Lee et al. 2004), whereas others failed or obtained low yields (Mareschi et al. 2001;

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Romanov et al. 2003). Recently, some groups have reported success in isolating and establishing MSCs cultures from Wharton’s jelly. (Mitchell et al. 2003; Romanov et al. 2003; Karahuseyinoglu et al. 2004; Wang et al. 2004; Lu-Lu et al. 2006; Chao et al. 2008). Secco et al. (2007) have recently reported that umbilical cord is rich in MSCs than UCB. Hence, MSCs from Wharton’s jelly was the choice of cells for isolation. In this report, we describe a simple, rapid, and reproducible method for simultaneous isolation of vascular endothelial and mesenchymal stem cells from a single umbilical cord. The work was conducted following the approval from the institutional ethical committee. After preinformed consents from the parents, umbilical cords were collected immediately after delivery from the local maternity hospital and were transported to lab in collection medium. Human umbilical vascular endothelial cells (hUVECs) were isolated as described by our group previously (UlrichMerzenich et al. 2002). The enzyme cocktail containing dispase II (Roche, Nutley, NJ) and collagenase type IV (Sigma Aldrich, St. Louis, MO) were used for the isolation of hUVECs from cord vein and human umbilical cord matrix stem cells (hUCMSCs) from Wharton’s jelly. Cord vein was filled with enzyme cocktail along with multiple site injection of enzyme cocktail in cord matrix. Whole cord was incubated at 37°C for 20 min, after which, ECs were seeded in a M199 and incubated in humidified atmosphere of 5% CO2 at 37°C. At the end of 4–6 h, the medium was changed to M199 containing 20% umbilical cord blood serum (UCBS), 2 mM L-glutamine, 5 ng/ml vascular endothelial growth factor (Chemicon International, Temecula, CA), 10 μg/ml heparin (Sigma Aldrich, St. Louis, MO), 100 U/ml penicillin, and 100 μg/ml streptomycin. After isolating ECs from umbilical cord, the rest of the cord was subjected to combination of collagenase and

dispase for initial 1 h followed by trypsin and EDTA for 30 min with continuous stirring. Since the enzymatic digestion is the crucial step for better yield of MSCs, we minimized the digestion time to one and half hour from the conventionally used overnight incubation (Wang et al. 2004). Finally, the cells were washed and cultured in Dulbeco’s modified Eagle’s medium with Ham’s F12 medium (1:1 v/v) (Invitrogen, Carlsbad, CA) supplemented with 10% UCBS (Phadnis et al. 2006) in 5% CO2 at 37°C incubator. ECs from vein attached well within a few hours to the fibronectin-coated surface and reached confluency within 6–8 days depending upon the initial seeding density. ECs were identified by their cobblestone morphology (Fig. 1A) and expression of von-Willibrand factor (Fig. 1B). Matrigel tube formation assay was used to assess the angiogenic potential (tube formation) of the isolated hUVEC. (Fig. 1C) Although ECs took longer time for attachment and proliferation, hUCMSCs could attach to tissue culture flasks within 24 h of culture. Floating cells were removed after 48 h. Homogenous fibroblast like cells were obtained (Fig. 2A) after reaching the confluency (Nanaev et al. 1997; Troyer and Weiss 2008). The cells were fixed in chilled 70% ethanol and incubated in mouse anti-human PEconjugated antibodies against CD33, CD34, CD 44, CD 45, CD 73, CD 90, CD 105, and CD117 (1:100 dil) for 1 h on ice (all the antibodies were purchased from Becton Dickinson, San Diego, CA). The cells were acquired using a flow cytometer laser 488 nm on BD FACS Vantage (Becton Dickinson, Franklin Lake, NJ), and data were analyzed using BD Cellquest Pro software. Expression of CD105 on hUMSCs was not seen in any of the samples analyzed. Deryl L. et. al. have recently mentioned in their mini review the presence of CD105 on mesenchymal stem cells derived from Wharton’s jelly. This could probably be a transient contamination of endothelial cells from the

Figure 1. Characterization of human umbilical vascular endothelial cells (hUVEC). Phase contrast image (A) cells are immunopositive for vonWillibrand factor (B) and form tube assay on Matrigel (C).

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Figure 2. Characterization of human umbilical cord matrix stem cells (hUCMSCs). Phase contrast image (A) Cells are immunopositive for smooth muscle actin (green) (B) and vimentin (red) (C). FACS analysis of hUCMSCs expressing CD 44, CD 73, CD 90, and CD117.

blood vessels which would exhibit CD105 on their surface (Dales et al. 2003; Fonsatti et al. 2003). Fluorescenceactivated cell sorting (FACS) analysis (Fig. 2D) showed that these cells were positive for CD44 (66.77%), CD90

(96.66%), CD73 (33.07%), and CD117 (48.98%) and were negative for CD33, CD34, CD45, and CD105. These cells also expressed cytoskeletal markers of mesenchymal origin, viz., smooth muscle actin and vimentin (Fig. 2B,C).

Figure 3. Differentiation potential of hUCMSCs. Accumulated neutral lipids stained with Oil Red O depicting adipogenesis (A). Deposition of sulfated proteoglycans stained with Safranin-O depicting chondrogenesis (B), whereas osteogenesis was observed by calcium-rich extracellular matrix stained with Alizarin Red S (C).

hUCMSCs exposed to neuronal differentiation medium, were immunopositive for Map2 (red) and NuN (green) (D). Lower panel indicates the negative control for the adipogenic (E), chondrogenic (F), osteogenic (G), and neuronal lineage (H).

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hUCMSCs could be subcultured for more than 15 passages and could be successfully revived after cryopreservation. Isolated hUCMSCs were further tested for their multipotent differentiation potential. When exposed to lineage-specific differentiation media (cocktails from Lonza, Basal, Switzerland), hUCMSCs could differentiate into adipogenic, chondrogenic, osteogenic, and neuronal lineages. Accumulation of neutral lipids within differentiated hUCMSCs was confirmed by staining these cells with Oil Red O (Fig. 3A). Chondrogenesis was demonstrated by sulfated proteoglycans stained with saffranin O (Fig. 3B) and osteogenesis by calcium deposition in extracellular matrix stained with Alizarin Red S (Fig. 3C). hUCMSCs exposed to neuronal differentiation medium expressed neuronal markers such as Map2 and NeuN (Fig. 3D), indicating their differentiation into neuronal lineage. We report here for the first time a two-step protocol for the simultaneous isolation of ECs and MSCs from a single umbilical cord. It was observed that UCBSsupplemented media further enhanced the growth and expansion of these cord-derived ECs and MSCs as compared to fetal calf serum (data not shown). The technique described here is highly reproducible, user friendly, and economical in terms of time and tissue requirement. The methodology of two-step isolation of ECs followed by MSCs permits easy dissection of the blood vessels, thus exposing Wharton’s jelly for enzyme treatment, reducing the digestion time without compromising the cell yield. The simultaneous isolation of ECs and MSCs from the same cord may also permit coculture directing the fate of MSCs towards endothelial cell lineage. Thus, the present study offers an economical and commercially viable option of endothelial cells as well as hUCMSCs for stem cell research and cell replacement therapy due to its noninvasive collection procedure, its being devoid of ethical concerns, and ease of isolation. Acknowledgments Authors wish to thank Director, NCCS for providing necessary facilities. Thanks are also due to Dr. Meeta Nakhare, Head of the Gynecology Department, Ratna Memorial Hospital, Pune, India for human umbilical cord samples and Department of Biotechnology, Government of India for providing financial support to carry out this work.

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