In vitro culture and differentiation of osteoblasts from human umbilical ...

Cell Tissue Bank (2010) 11:269–280 DOI 10.1007/s10561-009-9141-4

In vitro culture and differentiation of osteoblasts from human umbilical cord blood Tran Cong Toai Æ Huynh Duy Thao Æ Nguyen Phuong Thao Æ Ciro Gargiulo Æ Phan Kim Ngoc Æ Pham Hung Van Æ D. Michael Strong

Received: 29 March 2009 / Accepted: 14 June 2009 / Published online: 30 June 2009 Ó Springer Science+Business Media B.V. 2009

Abstract It is well accepted that human umbilical cord blood (UCB) is a source of mesenchymal stem cells (MSCs) which are able to differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. The aim of this study was to isolate MSCs from human UCB to determine their osteogenic potential by using different kinds of osteogenic medium. Eventually, only those MSCs cultured in osteogenic media enriched with vitamin D2 and FGF9, were positive for osteocalcin by RT-PCR. All these cells were positive for alizarin red, alkaline phosphatase and Von Kossa. The results obtained from RT-PCR have confirmed that osteogenesis is T. C. Toai H. D. Thao N. P. Thao C. Gargiulo Department of Histo-pathology, Embryology, Genetics and Biotechnology for Tissue Transplants, Pham Ngoc Thach Medical University, Ho Chi Minh City, Vietnam P. K. Ngoc University of Natural Science, Ho Chi Minh City, Vietnam P. H. Van The Central Lab of the University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam D. M. Strong (&) Department of Orthopaedics and Sports Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA e-mail: [email protected]

complete by expression of the osteocalcin marker. In conclusion, vitamin D2, at least in vitro, may replace vitamin D3 as an osteogenic stimulator factor for MSC differentiation. Keywords Mesenchymal stem cells (MSCs) Human umbilical cord (UCB) Bone marrow (BM) Major histocompatibility complex (MHC)

Introduction Multipotent mesenchymal stem cells Mesenchymal stem cells (MSCs) are a particular type of cell of embryonic mesodermal origin with a strong adherence ability and capable of differentiating into cells of different lineage tissues such as bone, cartilage, adipose tissues (Lee et al. 2004; Park et al. 2006; Bieback et al. 2004; Boissy et al. 2000; Chamberlain et al. 2007), and neural cells including astrocytes, neurons, hepatocystic and dermal tissue (Tse and Laughlin 2005; Chao et al. 2004; Koc and Lazarus 2001; Xu et al. 2004; Minguell et al. 2001; Song and Tuan 2004; Goodwin et al. 2001; Rosada et al. 2003; Kogler and Wernet 2006; Riordan et al. 2007; Kim et al. 2004; Jang et al. 2006; Kang et al. 2006; Van de Ven et al. 2007). Human MSCs have been isolated from various sources, such as adipose tissues, bone marrow (BM), umbilical cord blood (UCB), amniotic fluid, amniotic placenta, scalp tissue, amniotic membrane,

123

270

Cell Tissue Bank (2010) 11:269–280

and synovial tissue (Chao et al. 2004; Koc and Lazarus 2001; Xu et al. 2004; Minguell et al. 2001; Song and Tuan 2004; Goodwin et al. 2001; Rosada et al. 2003; Kogler and Wernet 2006; Riordan et al. 2007; Koblas et al. 2005). Functional analysis has confirmed that they share a core of gene expression profiles (Musina et al. 2007; Tsai et al. 2007; Reddi 2006; Tuan and Chen 2006; Chang et al. 2006; Maurice et al. 2007; de Bari and Dell’Accio 2007; Liu et al. 2007). MSC cultures in vitro are characterized by homogeneous colonies with a typical fibroblast spindle like shape (Lee et al. 2004, Park et al. 2006; Bieback et al. 2004; Minguell et al. 2001). For clinical applications, MSCs have been used to treat a wide variety of disorders, central nervous system pathologies, myocardial infarctions, hepatic failures, degenerative diseases such as diabetes or Crohn’s disease, in cell-based gene therapy as an alternative method in cartilage reconstruction and bone repair as in osteogenesis imperfecta (OI) (Horwitz et al. 2002), rheumatoid-arthritis (RA) and osteo-arthritis (OA) (Kogler and Wernet 2006; Riordan et al. 2007; Koblas et al. 2005; Kim et al. 2004; Reddi 2006; Tuan and Chen 2006; Waese and Kandel 2007; Lee et al. 2003; de Bari and Dell’Accio 2007). The MSC differentiation mechanism is a multi-step sequential process controlled by systemic and local factors, hormones, cytokines and genes. One of the most unique features of these groups of cells is their anti-inflammatory and immune-regulatory capacity (Tse and Laughlin 2005; Chao et al. 2004; Majhal et al. 2006; Koc and Lazarus 2001; Reiser et al. 2005).

Osteoblasts from human-UCB mesenchymal stem cells

MSCs from human UCB

Cell collection

It is widely recognized that human UCB is a good source of hematopoietic stem cells and has been used as a valid alternative source for hematopoietic stem cell transplantation (Lee et al. 2004; Park et al. 2006; Bieback et al. 2004; Chao et al. 2004; Goodwin et al. 2001; Rosada et al. 2003; Jang et al. 2006; Kang et al. 2006; Musina et al. 2007; Van de Ven et al. 2007; Maurice et al. 2007; Rebelatto et al. 2008). However, the UCB MSCs are able to switch into different cell phenotypes including osteoblasts–osteoclasts, chondrocytes, adipocytes, keratinocytes and neurons (Lee et al. 2004; Park et al. 2006; Bieback et al. 2004; Goodwin et al. 2001; Rosada et al. 2003; Kim et al. 2004; Jang et al. 2006; Kang et al. 2006; Musina et al. 2007; Van de Ven et al. 2007; Maurice et al. 2007).

Umbilical cord blood cells were collected from normal full-term and pre-term deliveries, following consent from the patient, and tested for HIV, HBV, HCV and syphilis by VDRL. The blood was collected with heparin anticoagulant, 15.000 UI/1 ml.

123

Bone tissue is a complex structure made of extracellular matrix, fibers, collagen type I, and other components such as glycoproteins which include osteonectin, osteopontin, bone sialoprotein, fibronectin and proteoglycans (Boissy et al. 2000; Aubin and Heersche 2000). This matrix becomes rigid due to the presence of hydroxypatite crystals which fill the interstitial spaces (Boissy et al. 2000). Osteoblasts are cells of mesenchymal origin present in endosteum, periosteum and BM and which form the core of bone matrix (Boissy et al. 2000; Reddi 2006; Stocum 2006; Aubin et al. 1995). The osteoblast formation, growth and maturation is a multi-sequential process controlled by local and systemic factors such as leptin, cytokines and different paracrine and autocrine factors (Boissy et al. 2000; Tuan and Chen 2006; Stocum 2006; Chang et al. 2006; Aubin and Heersche 2000; Aubin et al. 1995). In vivo the osteoblast phenotype shows a cuboidal form, a very intense activity of matrix deposit and presents a variety of protein expression such as collagen type I, osteopontin, alkaline phosphatase (AP) and osteocalcin typical of mature osteoblasts (Boissy et al. 2000). The aim of this study was to isolate MSCs from human UCB to determine their osteogenic potential by using different kinds of osteogenic media.

Materials and methods

Cell processing Processing UCB primary cells Mononuclear cells from UCB were isolated at a density of 1 9 106 cells/cm2, at room temperature, by using Ficoll-Paque (Amersham, Freiburg Germany) in a ratio of 1 part of Ficoll-Paque and 3 parts of blood and centrifuged (300g for 5 min). Cells were seeded


and cultured in flasks (Nunc, Wiesbaden, Germany) containing IMDM medium (IMDM, Gibco, Grand Island NY, USA) with 15% fetal bovine serum-FBS (Gibco USA). The total number of nucleated and viable cells was counted using Trypan Blue stain. Culture procedure for UCB mononuclear primary cells Mononuclear derived cells were cultured in IMDM medium plus 20% FBS and incubated at 37°C with 5% CO2. For the first week, medium was changed every 2 days and washed twice using PBS (buffer solution). Primary mononuclear cells began to attach at day 2, and were passed at day 15, once they reached a confluence of 70–80%. In each passage, cells were washed twice with PBS buffer solution and immersed in a solution of Trypsin–EDTA 2 ml (Gibco BRL USA) and incubated for 5 min at 37°C. After 5 min, 2 ml of IMDM plus 15% FBS v/v was added, cells were removed, transferred into a tube and centrifuged for 5 min at 200g. Suspended cells were removed, aspirated by pipette and seeded in new flasks at a density of 105/1 ml c.ca. Osteogenic medium composition for osteoblast differentiation Four different kinds of osteogenic media have been used to induce mesenchymal like cells from UCB differentiation into osteoblasts (Table 1). Medium n.1 composed of 65.2 ml of IMDM, 0.8 ml of penicillin– streptomycin, 15 ml FBS, 10.00 ml dexamethasone, 10.0 ml ascorbic acid, 2.0 ml b glycerol phosphate (all from Sigma-Aldrich USA). Medium n.2 composed of 42.2 ml of IMDM, 0.8 ml of penicillin–

271

streptomycin, 15 ml FBS, 10.00 ml dexamethasone, 10 ml ascorbic acid, 2.00 ml b glycerol phosphate (all from Sigma-Aldrich USA) and 20 ml vitamin D2 (Mekompharma-Ho Chi Minh city). Medium n.3 composed of 62 ml of IMDM, 0.8 ml of penicillin– streptomycin, 15 ml FBS, 10.00 ml dexamethasone, 10 ml ascorbic acid, 2.00 ml b glycerol phosphate (all from Sigma-Aldrich USA) and 0.2 ml FGF9. Medium n.4 composed of 42 ml of IMDM, 0.8 ml of penicillin–streptomycin, 15 ml FBS, 10.0 ml dexamethasone, 10.0 ml ascorbic acid, 2.00 ml b glycerol phosphate (all from Sigma-Aldrich USA) and 20 ml vitamin D2 and 0.20 ml FGF9. Cytochemical staining Mineral matrix deposits and bone nodules of both groups of osteoblasts, from UCB, was evaluated by staining cell cultures with alizarin red (AR), AP, Von Kossa (VK) and hematoxylin and eosin (HE). Alizarin red stain procedure The presence of calcium deposits were detected by washing cells with cold PBS and fixing them in 10% NFB-neutral formalin buffer solution for 30 min in a chemical hood. Cells were rinsed 3 times with distilled water and immersed in 2% (w/v) solution of AR for 30 s to 5 min. Cells were rinsed again with distilled water for 2–3 times and checked under an inverse microscope and photographed. Alkaline phosphatase stain procedure The presence of alkaline phosphates was detected by washing cells with cold PBS and fixing them in 10%

Table 1 Osteogenic medium Medium n.1

Medium n.2

Medium n.3

Medium n.4

65.2 ml of IMDM

42.2 ml of IMDM

62 ml of IMDM

42 ml of IMDM

0.8 ml penicillin–streptomycin




15 ml FBS

15 ml FBS

15 ml FBS

15 ml FBS

10.0 ml dexamethasone




10.0 ml ascorbic acid




2.00 ml b glycerol phosphate




20 ml vitamin D2

0.2 ml FGF9

20 ml vitamin D2 0.2 ml FGF9

123

272

NFB-neutral formalin buffer solution for 30 min and stained with a solution of naphtol As-MX-PO4 (Sigma) and Fast red violet LB salt (Sigma) for 45 min in the dark at room temperature. Cells were rinsed 3 times with distilled water and checked by inverse microscope and photographed. Von Kossa stain procedure The presence of calcium deposits were detected by washing cells with cold PBS and fixing them in 10% NFB-neutral formalin buffer solution for 30 min and stained with 2.5% silver nitrate (Merck-Germany) for 30 min in the dark. Cells were rinsed 3 times with distilled water, checked by inverse microscope and photographed. HE stain procedure Osteoblasts from UCB were stained using HE stain, a solution composed by hematoxylin 6.4 g, ammonium alum (60 g), ethanol (200 ml), glycerol (160 ml) and distilled water (640 ml) and, acidic alcohol composed of concentrated HCl (4 ml), 95% ethanol (396 ml); bluing agent composed of sodium bicarbonate (1 g) and distilled water (1 l). Samples have been overstained with hematoxylin for 5 min, washed in tap water for 2 min and re-stained for 10 s in acidic alcohol until they become reddish. The samples were rinsed in tap water and immersed in bluing agent (bicarbonate) for 2 min, rinsed with tap water, again dehydrated, cleared and covered with cytoseal. RT-PCR Total RNA extraction of the tested cell culture was carried-out including the positive control using the Trizol LS reagent (Invitrogen): (1) 150 ll of cell culture was added to one biopure eppendorf tube containing 450 ll Trizol LS (Invitrogen) and homogenized by pipetting up and down several times. (2) The homogenized solution was incubated at 30°C for 10 min. (3) 120 ll of chloroform was added, then rapidly and carefully shaken for 15 s; kept at 30°C for 10 min and centrifuged in a refrigerated centrifuge (2–8°C) at 13,000 rpm for 15 min. (4) 300 ll of the supernatant was carefully transferred into another biopure eppendorf tube while taken care not to disturb the interface, 300 ll isopropanol added, the

123


tube shaken gently up and down for a few seconds, then kept at 30°C for 10 min, centrifuged in the refrigerated centrifuge at 13,000 rpm for 20 min. (5) The supernatant was removed without losing the precipitated RNA (sometimes invisible) at the bottom or on one side of the bottom of the tube. (6) About 1 ml ethanol 80% was added without any shaking, then centrifuged in the refrigerated centrifuge at 8,000 rpm for 5 min; all of the supernatant was removed using a vacuum pump. (7) The pellet was dried at 55°C for 10–15 min, 40 ll Q water added to the dried pellet and then kept at 56°C for 10 min. The isolated RNA was stored at -20°C prior to RT-PCR. The cDNA synthesis was carried-out using the inscript cDNA synthesis kit (Biorad): (1) In one tube PCR 0.2, 1 ll reverse transcriptase was added, 4 ll RT mix, and 15 ll of the isolated RNA; gently mixed by pipetting up and down several times. (2) The cDNA synthesis carried-out in the thermal cycler by the following thermal cycle: 25°C/5 min, 42°C/30 min, 85°C/5 min, then kept at 4°C until PCR. Finally, the multiplex PCR was carried-out for osteocalcin and actin: (1) The PCR mix contained iTaq (Biorad) 1.25 l/reaction, iTaq PCR buffer 1X (Biorad), Mg?? 1.5 mM, dNTP 200 lM, osteocalcine forward primer (50 -CGC AGC CAC CGA GAC ACC AT-30 ) 25 pm/reaction, osteocalcine reverse primer (50 -GGG CAA GGG CAA GGG GAA GA-30 ) 25 pm/ reaction, actin forward primer (50 -CCA AGG CCA ACC GCG AGA AGA TGA C-30 ) 25 pm/reaction, actin reverse primer (50 -AGG GTA CAT GGT GGT GCC GCC AGA C-30 ) 25 pm/reaction; the volume of one PCR mix is 45 ll, left 5 ll for cDNA. (2) About 5 ll of the cDNA was added to the PCR mix, then the PCR was run in the iCycler (Biorad) with the following thermal cycle: 1 cycle: 95°C/5 min, 40 cycles: 94°C/30 s–55°C/30 s–72°C/1 min, 1 cycle: 72°C/10 min. (3) Results were read under agarose gel electrophoresis with #600 bps band for actin expression, and #400 bps band for osteocalcin expression.

Results In vitro culture of MSCs from UBC and their morphology To investigate and confirm the osteogenic potential of UCB derived stem cells low density mononuclear


273

cells were isolated from both original source, and cultured under appropriate conditions (Fig. 1). After 4–7 days cells started to form a few adherent, fusiform and elongated fibroblast like cells (Fig. 2). After 3 weeks these adherent cells started to form colonies generating a confluent monolayer of polyclonal cells, which were sub cultured and used in later experiments (Figs. 3, 4). These mesenchymal like cells were detected by inverse and electron microscope and were compared for morphology with MSCs from other studies (Fig. 4). These multipotent stem cells were induced to differentiate into osteoblasts, by using four different types of osteogenic media, that were named Medium n.1-2-3 and 4 (Tables 1, 2).

Table 2 Concentration gradient

Figs. 1–4 Mononuclear primary cells from human UCB after 2 days of culture, inverse microscope 9100. Primary cells from UCB start assuming fibroblast like shape, inverse microscope 9100. Mesenchymal like cells from UCB after

3 weeks stained by Giemsa, inverse microscope 9100. Mesenchymal like cells from UCB stained by Giemsa, inverse microscope 9100

Dexamethasone

10-7 M

b Glycerol phosphate

10 mM/ml

Ascorbic acid

50 ng/ml

FBS

15%

FGF9

10 ng/ml

Vitamin D2

10-7 M

Characteristic of adherent cells from MSCs culture from UCB To investigate and understanding the in vitro behavior of UCB stem cells, we have isolated mononuclear cells obtaining mesenchymal like cells

123

274


from human UCB. In the presence of osteogenic inducers human UCB-BM mesenchymal like cells started to change shape (between to 4 and 7 days), together with a continuous increase of AP and high presence of typical nodules of hydroxyapatite stained, respectively by AP (Fig. 5), AR (Fig. 6), VK (Fig. 7) and HE (Fig. 8). Although MSCs from human UCB were easier to culture and subculture, the initial period of isolating MSCs having them attached is critical with a very high rate of failure due to bacteria contamination or apoptosis. At the beginning cells started forming uniform agglomerates very similar to those from BM and, as already reported by others, the initial adherent

mesenchymal-like cells showed a spindle like shape which develop into multi-polar fibroblastoid cells. And in line with other results, over the first 2 weeks in culture (IMDM plus 15% FBS-data not shown) there was loss of the majority of cells, macrophages/ monocytes lineage first and hematopoietic later (Goodwin et al. 2001; Rosada et al. 2003). Cells were detected by inverse microscope and compared with other study results therefore identified as MSCs. UCB mesenchymal like cells have been passed up to the 12th generation with less than 1% loss over a period of 6 months. Some of the cultured mesenchymal like cells were induced into osteoblasts (Figs. 3, 4) and the remainder were

Figs. 5–8 Osteoblast like cells from UCB mesenchymal cells cultured in osteogenic medium enriched with vitamin D2 and FGF9 at day 25 stained with alkaline phosphatase, 9100. The osteogenic medium was composed IMDM, 15% FBS, Dexamethasone, Ascorbic acid, b Glycerol Phosphate enriched with vitamin D2 and FGF9 (all from Sigma-Aldrich USA). Osteoblast like cells from UCB mesenchymal cells, cultured

in osteogenic medium enriched with vitamin D2 and FGF9 stained at day 15 with alizarin red, 9100. Osteoblast like cells from UCB mesenchymal cells cultured in osteogenic medium enriched with vitamin D2 and FGF9 at day 20 stained with Von Kossa, 9100. Mesenchymal like cells from UCB cultured in osteogenic medium enriched with vitamin D2 and FGF9, at day 15 and stained with Giemsa, 9100

123


cryopreserved. Osteoblast like cells from UCB also showed a great capacity either in resistance or proliferation. At 4 weeks they start to produce a very strong and compact matrix, which was peeled off manually without using any enzymes, washed by PBS and chopped finely and sub-cultured again in different flasks. This new tissue started to attach a few hours later with an elevated presence of nodule and calcium deposits. Newly osteoblastic cell clusters started to form around it just a day later (data not shown). During 6 months of culture either MSCs or osteoblast like cells did not show any sign of apoptosis or decrease in proliferating activity. Osteoblast cultures have been cryopreserved and were successfully cultured again keeping intact their high proliferation capacity and could be passed several more times. Enrichment of vitamin D2 and FGF9 in classic osteogenic medium is essential for the expression of osteocalcin markers in osteoblast like cells from UCB by RT-PCR All Osteoblast like cells from UCB MSCs cultured in Medium n.1-2-3 and 4 (Table 1), were strongly positive for calcium matrix deposits by classical cytochemical staining like AR, AP and VK and positive for the expression of osteopontin marker by RT-PCR. However, the result was different for the expression of osteocalcin marker, which is considered a definitive osteogenic marker. Cell culture from normal osteogenic medium, Medium n.1 and Medium n.3 (Table 1) were negative for the expression of osteocalcin by RT-PCR. Osteogenesis was evaluated after a period of 2 weeks, 21 days, 3 and 6 months. The group of cells from Medium n.1 were tested for osteopontin and osteocalcin by RT-PCR at day 15, day 21, 2 and 6 months. Osteopontin expression was only positive at day 15 (Fig. 9), conversely osteocalcin was negative in all attempts. Only when osteogenic medium was enriched with vitamin D2 and FGF9, Medium n.4 (Table 1), at day 27, the expression of osteocalcin marker by RT-PCR (Figs. 10, 11) was observed. Osteogenic activity of UCB MSCs was induced without harvesting the cells at the second passage at 50% of confluence and old medium was simply replaced with a new osteogenic medium enriched with vitamin D2 and FGF9.

275

Discussion By definition, MSCs show some common specific features, morphologically they have fibroblast like shape, they are capable of self-renewal and differentiate into different connective tissue lineages of mesoderm such as bone, cartilage, fat and neurons (Lee et al. 2003, 2004; Park et al. 2006; Bieback et al. 2004; Tse and Laughlin 2005; Koc and Lazarus 2001; Xu et al. 2004; Minguell et al. 2001; Goodwin et al. 2001; Riordan et al. 2007; Tsai et al. 2007; Van de Ven et al. 2007; Reddi 2006; Tuan and Chen 2006; Chang et al. 2006), having an essential role in tissue repair and in growth process (Bieback et al. 2004). MSCs differentiation mechanism is a multi-step sequential process controlled by systemic and local factors, hormones, cytokines and genes and they can be relatively easily isolated from a variety of tissues and organs such as adipose tissues, BM, amniotic fluid, amniotic placenta scalp tissue, amniotic membrane and UCB (Lee et al. 2003, 2004; Park et al. 2006; Bieback et al. 2004; Tse and Laughlin 2005; Koc and Lazarus 2001; Xu et al. 2004; Minguell et al. 2001; Goodwin et al. 2001; Riordan et al. 2007; Koblas et al. 2005; Kim et al. 2004; Tsai et al. 2007; Van de Ven et al. 2007; Reddi 2006; Tuan and Chen 2006; Chang et al. 2006). There are fundamental qualitative and quantitative differences between MSCs and their progeny, which altogether depend on a great number of inter-related factors (Aubin and Heersche 2000; de Bari and Dell’Accio 2007). The clinical validity of UCB MSCs has been demonstrated in cases with or without HLA matching and have reported systemic injection of UCB MSCs (Tse and Laughlin 2005; Kogler and Wernet 2006; Waese and Kandel 2007). There is a beneficial effect from UCB mismatched cells even if they are cleared by the immune system, in a sense that they may play a positive role in inflammatory pathologies through activation of Th2 cells, as seen in mothers who received lymphocytes from their partners (Riordan et al. 2007). The MSCs differentiation mechanism involving osteogenesis includes a huge quantity of different factors (Reddi 2006). Bone is a very dynamic system which is constantly under a remodeling and renovation process (Waese and Kandel 2007). Osteogenesis presents similarities with embryonic development and morphogenesis (Reddi 2006). This happens due to a reciprocal involvement of

123

276


Figs. 9–11 The UCB-MSCs osteoblast phenotype was evaluated by the expression of osteopontin (15 days in osteomedium M1). The UCB-MSCs osteoblast phenotype was evaluated by the expression of osteocalcin marker (21 days in osteogenic medium enriched with vitamin D2 and FGF9) by using RTPCR following the manufacturer instructions. L1 is control

sample, L2 is Medium 1 (Table 1), L3 is Medium 2 (Table 1), L4 is Medium 3 (Table 1), L5 is Medium 4 (Table 1) and L6 is Human Osteoblasts. RT-PCR positive control for human osteoblasts confirming osteocalcin marker, L1 positive control osteocalcin and L2 human osteoblasts

systemic and local factors, controlled both transcriptionally and post-transcriptionally (Aubin and Heersche 2000), through hormones like leptin, or cytokines and different paracrine and autocrine factors, the disactivation of one of them may bring to a complete inhibition of any osteogenic activity and bone matrix production at any level (Boissy et al. 2000; Stocum 2006; Chang et al. 2006; Aubin and Heersche 2000; Lee et al. 2006; Blank et al. 2007; Yavrapoulou and Yovos 2007; Bodine 2008). Local control of bone homeostasis, generation, absorption and regeneration, is predominantly due to a synergic-

antagonist activity between the Wnt signaling pathway and the Notch signaling pathway, local transcriptor factors and some specific proteins which mediate the gene expression as histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Stocum 2006; Hilton et al. 2008; Long et al. 1995; Lee et al. 2006; Blank et al. 2007; Yavrapoulou and Yovos 2007). There is heterogeneity within the osteoblast family related to age and the state of bone, location and the presence of neighboring cells (Aubin and Heersche 2000). Therefore, the bone formation process is substantially a heterogeneous process

123


depending on the location (Aubin and Heersche 2000). The results of our study confirm the data of previous studies regarding the nature and behavior of MSCs from UCB and their differentiation into osteoblasts. We have isolated mononuclear cells from UCB and cultured them in medium containing IMDM plus 15% FBS (Figs. 1, 2, 3). After the critical first phase of seeding and culture of primary cells, we have successfully obtained without any substantial loss (less than 1%), over 40 cultures of MSCs up to the 12th generation (12 passages) (Fig. 4), and osteoblasts from a single UCB primary cell sample. Eventually, either osteoblasts from UCB were positive for traditional stains such as AR (Fig. 6), AP (Fig. 5), VK (Fig. 7). In line with other results, MSCs and osteoblasts from human UCB have shown a longer lifespan in vitro, surviving well over 6 months without any sign of apoptosis (Lee et al. 2004; Park et al. 2006, 2007; Bieback et al. 2004; Goodwin et al. 2001; Rosada et al. 2003; Kogler and Wernet 2006; Kim et al. 2004; Jang et al. 2006; Kang et al. 2006; Musina et al. 2007; Van de Ven et al. 2007; Chang et al. 2006; Kalmoz et al. 2006). Furthermore, both osteoblasts and MSCs from UCB have been safely cryopreserved and stored for long period without losing any of their intrinsic growth and proliferation capacity. We assume this has to be related to their immature state and their longer telomeres (Chang et al. 2006). Osteoblasts from human UCB showed a remarkable capacity of adherence exhibiting a stronger AP activity, with a markedly high accumulation of mineral nodules. With osteoblasts from UCB MSCs culture, it was noted that, after a few weeks of culture, cells start to form dense colonies and about a month later a tight compact layer. This tissue was removed manually without using any enzymes and sub-cultured again. Strongly adherent, it attached a few hours later. While 12 h later, the presence of typical nodules together with newly osteoblasts formation were observed all around it; 36 h later formation of mineralizing colonies and calcium deposit were detected. However, though all osteoblast cultures from UCB cultured in four different kinds of media (Table 1) were strongly positive for calcium deposit by cytochemical stain such as AR, AP and VK and were morphologically identical to osteoblast cells from other studies, none of the

277

cultures in osteogenic Medium n.1, 2 and 3 (Table 1) gave positive results for osteocalcin marker by RT-PCR. This result may be explained by the fact that expression of osteocalcin is not correlated with the degree of cytochemical stains like AR, AP and VK (Sudo et al. 2007). Therefore, an alternative solution was adopted suggested by Maurice et al. (2007) who asserted that the enrichment of vitamin D3 and FGF9 in their osteogenic medium was essential for the positive expression of osteocalcin. This finding was corroborated by other studies which stated that in vitro differentiation of embryonic stem cells (ES) and MSCs need a few important factors, among them are ascorbic acid, dexamethasone and 1,25 dihydroxyvitamin D3, essential elements for matrix deposition and mineralization (Purpura et al. 2004; Duplomb et al. 2007; Jono et al. 1998; Long et al. 1995). Of note, in a study on vascular calcification, where the atherosclerotic plaque formation has been considered a similar process to osteogenesis, 1,25 dihydroxyvitamin D3 is considered the main factor responsible for calcium influx into vascular smooth muscle cells and the great inhibitor of their proliferation (Jono et al. 1998). However, since vitamin D3 is not available in Vietnam and the costs to receive it are notably high we opted for a suitable replacement of vitamin D3. Comparative studies on the use of vitamin D3 and D2 demonstrated that vitamin D2 and D3 have similar effects (Holick et al. 2008). Thus eventually, vitamin D2 and FGF9 were added into the osteogenic medium and the results were that only osteoblasts in osteogenic medium enriched with vitamin D2 and FGF9 were positive for osteocalcin marker tested by RT-PCR (Fig. 3). Data which confirm that the combination of vitamin D2 and FGF9 gave more reliable results obtaining a stronger and more complete osteogenic differentiation of MSCs. Altogether these results eventually indicate that MSCs may be induced to differentiate in very early stage without the need of harvesting them. Vitamin D2, like vitamin D3, shows the same induction capacity and is essential for the expression of osteocalcin. Conversely, osteopontin expression does not require any use of additional growth factors such as vitamin D or FGF9, cells simply produce it within a period of two three weeks. In addition, following published works, natural coral scaffolds were prepared and seeded with

123

278

osteoblast lines from UCB (Chen et al. 2002, 2004). These cells successfully seeded onto coral scaffolds and expanded after 15 days.

Conclusion Umbilical cord blood MSCs from our study showed homogeneous characteristics and morphology. The results obtained from RT-PCR have confirmed that osteogenesis is complete by the expression of osteocalcin marker only when osteogenic medium is enriched with two additional factors, vitamin D2 and FGF9. Finally, our results indicate that vitamin D2 at least in vitro may replace vitamin D3 as an osteogenic stimulator factor for MSC differentiation. In conclusion one fact is well consolidated, the future of the effective clinical use of stem cells still requires more research. As some authors diligently warn several crucial issues need to be solved before considering their definitive use in clinical treatments for human diseases, to ensure that it is clear what will happen to stem cells once they are transfused in a new environment and their role either as healing factors or etiopathogenic agents (de Bari and Dell’Accio 2007; de Winter 2003).

References Aubin JE, Heersche JN (2000) Osteoprogenitor cell differentiation to mature bone forming osteoblasts. Drug Dev Res 49:206–215. doi:10.1002/(SICI)1098-2299(200003)49:3 \206::AID-DDR11[3.0.CO;2-G Aubin JE, Malaval F, Gupta AK (1995) Osteoblasts and chondroblasts differentiation. Bone 17(2):77–83. doi:10.1016/ 8756-3282(95)00183-E Bieback K, Kern S, Kutler H, Eichler H (2004) Critical parameters for isolation of mesenchymal stem cells from umbilical cord. Stem Cells 22:625–634. doi:10.1634/stemcells. 22-4-625 Alpha Med Press Blank U, Karlsson G, Karlsson S (2007) Signaling pathway governing stem cell fate. Blood 111(2):492–503. doi: 10.1182/blood-2007-07-075168 Bodine PVN (2008) Wnt signaling control of bone cell apoptosis. Cell Res 18:248–253. doi:10.1038/cr.2008.13 Boissy P, Malaval L, Jurdic P (2000) Osteoblasts et osteoclastes une cooperation exemplaire entre cellules mesenchymateuses et cellules hematopoietiques. HematologieMontrouge 6(1):6–16 Chamberlain G, Fox J, Ashton B, Middleton J (2007) Mesenchymal stem cells: their phenotype, differentiation

123

Cell Tissue Bank (2010) 11:269–280 capacity, immunological features and potential for homing. Stem Cells 25:2739–2749. doi:10.1634/stemcells. 2007-0197 Chang YJ, Shih DTB, Tseng CP, Hsieh TB, Lee DC, Hwang SM (2006) Disparate mesenchymal lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord. Stem Cells 24:679–685. doi:10.1634/stem cells.2004-0308 Chao NJ, Emerson SG, Weinberg KI (2004) Stem cell transplantation. Hematology (Am Soc Hematol Educ Program) 354–371. doi:10.1182/asheducation-2004.1.354 Chen F, Tao K, Mao T, Chen S, Ding G, Gu X (2002) Bone graft in the shape of human mandibular condyle reconstruction via seeding marrow-derived osteoblasts into porous coral in a nude mice model. Am Assoc Oral Maxillofac Surg 60: 1155–1158 Chen F, Chen S, Tao K, Feng X, Liu Y, Lei D, Mao T (2004) Marrow derived osteoblasts seed into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits. Br J Oral Maxillofac Surg 42:532–537 de Bari C, Dell’Accio F (2007) Mesenchymal stem cells in rheumatology: a regenerative approach to joint repair. Clin Sci 113:339–348. doi:10.1042/CS20070126 de Winter EA (2003) What is the future of stem cells? Cytotechnology 43:133–138. doi:10.1023/A:1024874706356 Duplomb L, Dagouassat M, Jourdon P, Heymann D (2007) Embryonic stem cells: a new tool to study osteoblast and osteoclasts differentiation. Stem Cells 25:544–552. doi: 10.1634/stemcells.2006-0395 Goodwin HS, Bicknese AR, Chien SN, Bogucki BD, Olivier DA, Qinn CO, Wall DA (2001) Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 7:581–588. doi:10.1053/bbmt.2001. v7.pm11760145 Hilton MJ, Tu X, Wu X, Bai S, Zhao H, Kobayashi T, Kronenberg HM, Teitelbaum S, Ross P, Kopan R, Long F (2008) Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med 14(3):306–312. doi:10.1038/nm1716 Holick MF, Biancuzzo RM, Chen TC, Klein EK, Young A, Bibuld D, Reitz R, Salameh W, Ameri A, Tannenbaum AD (2008) Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab 3:677–681 Horwitz EM, Gordon PL, Koo W, Marx JC, Neel MD, McNall RY, Muul L, Hofman T (2002) Isolated allogeneic bone marrow derived mesenchymal stem cells engraft and stimulate growth in children with osteogenesis imperfecta: implication for cell therapy of bone. Proc Natl Acad Sci USA 99(13):8932–8937. doi:10.1073/pnas.132252399 Jang YK, Jung DH, Jung MH, Kim DH, Yoo KH, Sung KW, Oh W, Yang YS, Yang SE (2006) Mesenchymal stem cells feed layer from human umbilical cord blood for ex vivo expanded growth and proliferation of hematopoietic progenitor cells. Ann Hematol 85:212–225. doi:10.1007/ s00277-005-0047-3 Jono S, Nishizawa Y, Shioi A, Morii H (1998) 1,25 dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid

Cell Tissue Bank (2010) 11:269–280 hormone-related peptide. J Am Heart Assoc Circ 98: 1302–1306 Kalmoz LP, Kolbus A, Wick N, Mazal PR, Eisenbock B, Burjak S, Meissl G (2006) Cultured human epithelium: human umbilical cord blood stem cells differentiate into keratinocytes. Burns 32:16–19. doi:10.1016/j.burns.2005. 08.020 Kang XQ, Zang WJ, Bao LJ, Li DL, Xu XL, Yu XJ (2006) Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro. Cell Biol Int 30:569–575. doi:10.1016/j.cellbi.2006.02.007 Kim JW, Kim SY, Park SY, Kim YM, Kim JM, Lee MH, Ryu HM (2004) Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol 83:733–738. doi:10.1007/ s00277-004-0918-z Koblas T, Harman SM, Saudek F (2005) The application of umbilical cord cells in the treatment of diabetes mellitus. Rev Diabet Stud 2:228–234. doi:10.1900/RDS.2005.2.228 Koc O, Lazarus HM (2001) Mesenchymal stem cells: heading into the clinic. Bone Marrow Transplant 27:235–239. doi: 10.1038/sj.bmt.1702791 Kogler G, Wernet P (2006) Pluripotent stem cells from umbilical cord; stem cell transplantation. Biol Process Ther 7:3–86 Lee HS, Huang GT, Chiou LL, Chen MH, Hsieh CH, Jiang CC (2003) Multipotent mesenchymal stem cells from bone marrow near site of osteonecrosis. Stem Cells 21:190– 199. doi:10.1634/stemcells.21-2-190 Lee OK, Kuo TK, Chen WM, Lee KD, Hsien SL, Chen TH (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103(5):1669–1675. doi: 10.1182/blood-2003-05-1670 Lee HW, Suh JH, Kim AY, Lee YS, Park SY, Kim JB (2006) Histone deacetylase 1-mediated histone modification regulates osteoblasts differentiation. Mol Endo 20(10): 2432–2443 Liu TM, Martina M, Hutmacher DW, Po Hui JH, Lee EH, Lim B (2007) Identification of common pathways mediating differentiation of bone marrow and adipose tissue derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells 25(3):750–760. doi:10.1634/stemcells. 2006-0394 Long MW, Robinson JA, Ashcraft EA, Mann KG (1995) Regulation of human bone marrow derived osteoprogenitor cells by osteogenic growth factors. J Clin Invest 95:881–887. doi:10.1172/JCI117738 Majhal NS, Weisdorf DJ, Wagner JE, Defor TE, Brunstein CG, Burns LJ (2006) Comparable results of umbilical cord blood and HLA matched sibling donor hematopietic stem cell transplant after reduced-intensity preparative regimen for advanced Hodgikin’s lymphoma. Blood 107(9):3804– 3807. doi:10.1182/blood-2005-09-3827 Maurice S, Srouji S, Livne E (2007) Isolation of progenitor cells from cord blood using adhesion matrices. Cytotechnology 54(2):121–133 Minguell JJ, Erices A, Conget P (2001) Mesenchymal stem cells. Soc Exp Biol Med 226:507–520 Musina RA, Bekchanova ES, Belyaskii AV, Grinenko TS, Sukhikh GT (2007) Umbilical cord blood mesenchymal stem cells. Bull Exp Biol Med 143(1):15–20. doi: 10.1007/s10517-007-0032-z

279 Park KS, Lee YS, Kang KS (2006) In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood. J Vet Sci 7(4):343–348 Park KS, Jung KH, Kim SH, Choi MR, Kim Y, Chai YG (2007) Functional expression of ion channels in mesenchymal stem cells derived from umbilical cord vein. Stem Cells 25(8):2044–2052. doi:10.1634/stemcells.2006-0735 Purpura KA, Aubin JE, Zandstra PW (2004) Sustained in vitro expansion of bone progenitors is cell density dependent. Stem Cells 22:39–50. doi:10.1634/stemcells.22-1-39 Rebelatto CK, Aguiar AM, Moreta˜o MP, Senegaglia AC, Hansen P, Barchiki F, Oliveira J, Martins J, Kuligovski C, Mansur F, Christofis A, Amaral VF, Brofman PS, Goldenberg S, Nakao LS, Correa A (2008) Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med 233:901–913. doi:10.3181/0712-RM-356 Reddi AH (2006) Bone regeneration. In: Battler A, Leor J (eds) Stem cell and gene-based therapy: frontiers in regenerative medicine. Springer-Verlag, New York, pp 195–199 (13) Reiser J, Zhang XY, Hemenway CS, Mondal D, Pradhan L, La Russa VF (2005) Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther 5(12):1571–1584. doi: 10.1517/14712598.5.12.1571 Riordan NH, Chan K, Marleau AM (2007) TE Ichim; cord blood in regenerative medicine: do we need immune suppression? J Transl Med 5(8):1–9 Rosada C, Justensen J, Melsvik D, Ebbesen P, Kassem M (2003) The human umbilical cord blood: a potential source for osteoblast progenitor cells. Calcif Tissue Int 72:135–142. doi:10.1007/s00223-002-2002-9 Song L, Tuan RS (2004) Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J 18:980–985 Stocum DL (2006) Regenerative biology and medicine. Elsevier, Boston, pp 229–237 Sudo K, Kanno M, Miharada K, Ogawa S, Hiroyama T, Saijo K, Nakamura Y (2007) Mesenchyma progenitors are able to differentiate into osteogenic, chondrogenic, and/or adipogenic cells in vitro are present in most primary fibroblast-like cell population. Stem Cells 25:1610–1617. doi:10.1634/stemcells.2006-0504 Tsai MS, Hwang SM, Chen KD, Lee YS, Hsu LW, Chang YJ, Wang CN, Peng HH, Chang YL, Chao AS, Chang SD, Lee KD, Wang TH, Wang HS, Soong YK (2007) Functional network analysis on the transcriptomes of mesenchymal stem cells derived from amniotic fluid, amniotic membrane, cord blood and bone marrow. Stem Cells Express 25(10):2511–2523 Tse W, Laughlin MJ (2005) Umbilical cord transplantation: a new alternative option. J Hematol 37:7–383 Tuan RS, Chen FH (2006) Cartilage. In: Battler A, Leor J (eds) Stem cell and gene-based therapy: frontiers in regenerative medicine. Springer-Verlag, New York, pp 179–189 (12) Van de Ven C, Collins D, Bradley B, Morris E, Cairo MS (2007) The potential of umbilical cord blood multipotent stem cells for nonhematopoietic tissue and cell regeneration. Exp Hematol 35:1753–1765. doi:10.1016/j.exphem. 2007.08.017

123

280 Waese EY, Kandel R (2007) Application of stem cells in bone repair. Skeletal Radiol 37(7):601–608 Xu W, Zhang X, Qian H, Zhu W, Sun X, Hu J, Zhou H, Chen Y (2004) Mesenchymal stem cells from adult bone marrow differentiate into a cardiomyocyte phenotype in vitro. Soc Exp Biol Med 229:623–631

123

Cell Tissue Bank (2010) 11:269–280 Yavrapoulou MP, Yovos JG (2007) The role of the Wnt signaling pathway in osteoblast commitment and differentiation. Hormones 6(4):279–294