Cell Tissue Bank (2011) 12:125–133 DOI 10.1007/s10561-010-9174-8 In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture Tran Cong Toai • Huynh Duy Thao • Ciro Gargiulo Nguyen Phuong Thao • Tran Thi Thanh Thuy • Huynh Minh Tuan • Nguyen Thanh Tung • Luis Filgueira • D Micheal Strong • Received: January 2010 / Accepted: March 2010 / Published online: 27 March 2010 Ó Springer Science+Business Media B.V 2010 Abstract There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of days and tested by immunostain p63 and K1-K10 Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro Keywords Mesenchymal stem cell Á UCB Á Keratinocyte culture Á Cell culture Introduction T C Toai (&) Á H D Thao Á N P Thao Á T T T Thuy Á H M Tuan Department of Histo-pathology, Embryology, Genetics and Biotechnology for Tissue Transplants, Pham Ngoc Thach Medical University, Ho Chi Minh City, Vietnam e-mail: toaiphd@yahoo.com C Gargiulo Á L Filgueira University of Western Australia School of Anatomy and Human Biology, Crawley, WA, Australia D M Strong Department of Orthopaedics and Sport Medicine, University of Washington School of Medicine, Seattle, WA, USA N T Tung Department of Pathology, Children No Hospital in HCMC, Ho Chi Minh City, Vietnam MSCs from human-UCB Human UCB is a significant source of hematopoietic stem cells and has been considered as a valid alternative for hematopoietic stem cell transplantation (Toai et al 2009; Lee et al 2004; Park et al 2006; Van de Ven et al 2007; Maurice et al 2007; Musina et al 2007; Sasaki et al 2008) MSCs from hUCB have been used in a wide range of diseases such as liver disorders, myocardial infarction, central nervous system condition or in degenerative pathologies such as diabetes, Crohn’s disease, osteogenesis imperfect (OI), rheumatoid arthritis (RA) and osteoarthritis (OA) (Toai et al 2009; Lee et al 2004; 123 126 Riordan et al 2007; Kogler and Wernet 2006; Kim et al 2004; Reddi 2007; Koblas et al 2005; De Bari and Dell’Accio 2007; Tuan and Chen 2006; Waese and Kandel 2007; Park et al 2006) The most valuable potential of MSCs is their ability to switch into different cell phenotypes such as osteocytes, chondrocytes, adipocytes, hepatocytes, neurons, myocytes and keratinocytes with a great immunemodulatory and anti-inflammatory capacity that make them a tool for clinical applications (Toai et al 2009; Lee et al 2004; Goodwin et al 2001; Chamberlain et al 2007; Kim et al 2004; Bieback et al 2004; Musina et al 2007; Jang et al 2006; Rosada et al 2003; Van de Ven et al 2007; Maurice et al 2007; Tse and Laughlin 2005; Koc and Lazarus 2001; Chao et al 2004; Majhal et al 2006; Sasaki et al 2008; Stocum 2006) The skin is a barrier to the outside elements, temperature loss, pathogens and trauma (Markowicz et al 2005) The use of skin substitutes for skin replace in cases of burns and ulcers is a developing field, however nothing works better than patient’s own skin (Markowicz et al 2005) The inconvenience of using bio-engineered materials for skin graft replacement is connected to the allogeneic origin of these cells hence these bio-products can only be used for wound coverage and not as a graft for tissue substitution (Markowicz et al 2005) Nevertheless, many studies have confirmed a conspicuous advantage of UCB engraftment related with a very low rate of transplant mortality and no increase of rejection or graft versus host disease (GVHD) due to a high rate of tolerance across or HLA-A, B and DR mismatches and a lower risk of infectious disease transmission (Van de Ven et al 2007; Toai et al 2009; Lee et al 2004; Riordan et al 2007; Kogler and Wernet 2006; Tse and Laughlin 2005) The idea to obtain keratinocytes from hUCB MSCs is mainly due to their particular ability to differentiate into different cell phenotypes and their immune-modulatory and anti-inflammatory nature that is crucial in the case of allograft procedures for skin regeneration (Van de Ven et al 2007; Toai et al 2009; Lee et al 2004; Riordan et al 2007; Kogler and Wernet 2006; Tse and Laughlin 2005; Kamolz et al 2006) Human UCB have been shown to have a very limited number of graft lymphocytes and hUCB MSCs are able to secrete inhibitory cytokines such as IL10 and TFG-b whilst maintaining the ability of presenting antigens 123 Cell Tissue Bank (2011) 12:125–133 to T cells, a condition that eventually confirm a tolerogenic antigen capacity of this group of stem cells (Toai et al 2009; Lee et al 2004; Riordan et al 2007) Moreover, UCB T cells are distinctively CD45RA? with low intensity of activation markers, both of which are related with naăve Th0 phenotype that show a restricted response triggered by recipient alloantigens (Tse and Laughlin 2005) When isolated CD34? cells from allogeneic cord blood were inserted in an autologous fibrin glue of patients with non-healing wounds, it was noted that a significant wound repair was achieved without any sign of GVHD from to months subsequent to the procedure (Riordan et al 2007) Then again, Kamolz et al successfully used male hUCB stem cells together with skin from female donors to obtain keratinocytes in vitro Using PCR they confirmed the presence of keratinocytes among the hUCB stem cell population and by FISH histochemistry they revealed Y-positive cells within the keratinocytes layer In addition, they detected hUCB cells among all layers of cultured epidermis (Kamolz et al 2006) Overall, these data eventually confirm the capacity of hUCB stem cells as a budding resource for cultivating human epithelium under vitro conditions Therefore, this study was undertaken to demonstrate the ability of hUCB to be directly influenced to produce keratinocytes in vitro Materials and methods Cell collection Umbilical cord blood cells were collected and isolated from consenting patients from normal full term and pre-term deliveries The material was serology tested for HIV, HBV, HCV and syphilis by VDRL The blood was collected with heparin anticoagulant, 15000UI/1 ml Cell processing Processing UCB primary cells Mononuclear cells from hUCB were isolated at a density of 106 at room temperature using FicollPaque (Amersham, Freiburg-Germany) in a ratio of part of Ficoll-Paque and parts of blood and Cell Tissue Bank (2011) 12:125–133 centrifuged, 300g for Cells were collected and seeded in flasks (Nunc, Wiesbaden-Germany) containing 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 hUCB mononuclear primary cells Mononuclear derived cells were incubated at 37°C with 5% CO2 During the first week, medium was changed every days and cells washed twice by buffer solution (PBS) Primary mononuclear cells began to attach at day 2, cells were passed at day 15 at 70–80% confluence After each passage, cells were washed twice with PBS and immersed in a ml solution of Trypsin–EDTA (Gibco, Brl-USA) and incubated for at 37°C with 5% CO2 After min, ml of IMDM plus 15% FBS was added, cells were removed, aspired and transferred in a tube for centrifugation for at 200g Suspended cells were removed and seeded in new flasks at a density of 105/1 ml c.ca Culture procedure for direct keratinocytes differentiation MSCs were induced to differentiate into keratinocytes by seeding them into a medium composed of a combination of PCM plus SFM for a period of 7, 14 and 17 days Human UCB MSCs were subcultured times, after which medium was changed and MSCs were directly induced to differentiate by using a keratinocytes medium composed of PCM plus SFM in a ratio of 1:9 At the day cells were trypsinized (using Trypsin–EDTA) and collected for K1-10 immune-stain At the 14th–17th days, other cell samples were collected and stained with p63 Skin collection for keratinocyte staining with p63 antibody A serum collected from previous consented patient’s was serology tested for HIV, HBV, HCV and syphilis by VDRL The sample was collected with heparin anticoagulant, 15000UI/1 ml 127 Keratinocyte medium composition Keratinocyte medium is composed of part of PCM and parts of SFM PCM medium DMEM medium (Gibco Grand Island NY-USA), HEPES M (Sigma Ultra), FBS 15%, EGF 100 mg/ ml (Gibco Invitrogen Corporation), Cholera toxin 10-7M (List biological Laboratories Inc.), Hydrocortisone 0,1 mg/ml (Westcort USA), Penicillin/Streptomycin 2009 Defined keratinocyte-SFM Medium (Gibco Invitrogen Corporation) Immunohistochemical stain P63 histochemical stain procedure Immunohistochemical staining was performed according to standard procedures Sample slides and positive control were stored in an incubator at 37°C overnight Slides and control were deparaffinerized by xylene twice for and washed by alcohol 100, 90, 80%, respectively for each passage For antigen retrieval, slides were immersed in buffer solution at pH and steamed in microwave oven for 25 s (S2368-Dako) Endogenous peroxidase activity was blocked by PBS and incubated in 3% hydrogen peroxide (H2O2) for 10 The antibody used for p63 was obtained commercially from Neomarkers (Fremont, CA, USA) and was used at a 1:25 dilution The detection step was performed using an LSAB2 System—HPR- Dako ? kit (DAKO; Carpinteria, CA) as chromogen at 1:20 dilution rate for 20 Samples were counterstained with streptavidin HRP and hematoxylin (Biomeda-M10) K1-10 Keratinocyte immunostain fluorescence staining procedures The cells were fixed in 1% paraformaldehyde in culture medium before they were mechanically detached and spun onto slides using a Shandon centrifuge (4 min, 600 rpm) Thereafter the cells were treated for with 0.1% triton 1009 (ICN Biomedicals; Aurora, OH) in PBS, before staining with the mouse anti-human keratin 1/10 monoclonal 123 128 Cell Tissue Bank (2011) 12:125–133 antibody (1ug/ml, CBL266, Chemicon/Millipore, North Ryde, NSW, Australia) for h, followed by AlexaFluor488 donkey-anti mouse (1:100, Molecular Probes/Invitrogen, Mulgrave, Victoria, Australia) for h The nuclei were counterstained with DAPI (1ug/ ml, Roche Diagnostics, Mannheim, Germany) and the F-actin filaments with AlexaFluor546 labeled phalloidine (0.3 Units/ml, Molecular Probes/Invitrogen) Control staining was done similarly without the primary antibody The cells were mounted with Dako fluorescence mounting medium (DakoCytomation; Carpinteria, CA) The specimens were analyzed and documented with a Nikon Eclipse 90i microscope with fluorescence and conventional setting, including corresponding digital cameras and imaging software Fig Human UCB monoclonal primary stem cells after days of culture in IMDM ?10% FBS, inverse microscope 9100 Results In vitro culture of keratinocytes from MSCs from hUCB and their morphology To confirm the keratinocytic potential of hUCB derived stem cells, low density mononuclear cells were isolated from the original source and cultured under proper condition with IMDM plus 15% FBS (Figs 1, 2, 3, 4) In line with other studies, after a few days of culture, mononuclear cells started to form clusters of adherent cells with typical fusiform and elongated fibroblast shape (Figs 3, 4) After weeks cells reached 70–80% of confluence forming a dense monolayer of polyclonal cells, at this stage cells were trypsinized and cultured for a total of times At the 3rd passage the old medium was removed and a new keratinocyte medium was added composed of PCM plus SFM Cells started to change shape at day assuming a more round-cuboidal conformation typical of keratinocyte like cells (Figs 5, 6, 7, 8, 9, 10) Cells were constantly monitored by inverse microscope and compared with cells from different studies (data not shown) At day 7, part of samples were selected and tested for immunohistochemical staining to confirm the presence of K1-10 (Figs 15, 16) At day 14 and 17, the rest of samples were collected and tested for p63 antibody reactivity (Figs 11, 12, 13, 14) A sample of human skin was stained with p63 antibody by immunohistochemical staining as a positive control (Fig 17) 123 Fig Primary cells from hUCB after days of culture start assuming fibroblast like shape, inverse microscope 9100 Discussion By definition, MSCs show regular features includinga fibroblast like morphology, a high rate of selfrenewal aptitude, an unusual capacity of differentiating into different cell phenotypes and the ability to play a prominent role in tissue repair and the growth process (Toai et al 2009; Lee et al 2004; Bieback et al 2004; Minguell et al 2001; Reddi 2007; Sasaki et al 2008) Because MSCs, in our experience, qualify by these criteria, we named these cells mesenchymal stem cells in the current article Cell Tissue Bank (2011) 12:125–133 129 Fig MSCs from UCB in keratinocyte medium culture after days (2009) Fig After days of culture cells assumed a complete mesenchymal morphology, inverse microscope 9100 Fig MSCs from UCB in keratinocyte medium culture after days (1009) Fig MSCs at day 10 start forming a compact and dens monolayer the confluence is nearly 60–70%, inverse microscope 9100 A number of attempts have been performed to use MSCs in clinical trials in order to regenerate tissues in spinal cord injuries, myocardial infarction, bone diseases, neurological diseases and skin (Mazzini et al 2006; Chernykh et al 2006; Waese and Kandel 2007; Kamolz et al 2006) Meanwhile, there have been many efforts to attain and culture human keratinocytes for clinical purposes including: for skin replacement or wound repair for use as bio-material or skin substitutes from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE) from skin specimens in media with feeder layers and from suction blister epidermal roofs in serum-free culture (Sasaki et al 2008; Kamolz et al 2006) However, only a few have directly obtained keratinocyte cultures from hUCB MSCs without the support of any exogenous feed layers in vitro The results from those studies eventually validate the possibility of using these cells as tools in skin regeneration therapy in vivo (Kamolz et al 2006; Sasaki et al 2008) The microenvironment is of great importance for the recruitment of circulating MSCs at the affected site The inflammation mechanism plays a crucial role in the wound healing process because of the accrual of multiple inflammatory factors and cells which promote tissue recovery and the regeneration 123 130 Fig Keratinocyte medium is composed of FSM and PCM, ratio 1:9, the MSCs started changing shape at 3rd day of culture gradually assuming a typical round cuboidal keratinocyte shape MSCs were induced without harvest procedure the old medium was changed on situ with the new one Cell Tissue Bank (2011) 12:125–133 Fig Keratinocytes monolayer after 17 days of culture 9200 Fig 10 The MSCs completed their differentiation in 17 days c.ca, 9200, once the cultures reached the 70–80% confluence they were collected and tested for immunohistochemical stain for p63 and K1-10 Fig MSCs, control group, 80% confluence, 15 days 9100 MSCs in keratinocyte medium after weeks culture c.ca It’s possible to visualize the presence of round shape keratinocyte cells 9100 process by refilling of cells and extracellular components (Sasaki et al 2008; Kamolz et al 2006) Keratinocytes at the wound site express Chemokine (C–C motif) ligand 21 and secondary lymphoid-tissue chemokine (SLC/CCL21) inducing a high presence of MSCs that eventually promote the repair by transdifferentiation into multiple skin cells (Sasaki et al 2008) This mechanism takes place because MSCs express several chemokine receptors including CCR7 which is a receptor of SLC/CCL21 that enhances the recruitment of MSCs in loco (Sasaki et al 2008) 123 Fig 11 keratinocytes immunohistochemical stain with p63 at day 14, red indicates presence of p63 9200 Cell Tissue Bank (2011) 12:125–133 131 Fig 12 Control group stained with Giemsa 9200 Fig 15 Keratinocytes stained for keratin 1/10, green color, nuclei blue color, by electronic microscope Fig 13 Keratinocyte immunochemical stain for p63 at day 14, red indicates presence of p63 and blue indicates the nucleus, inverse microscope 9400 Fig 16 Control group stain, electronic microscope Keratinocytes at day culture were collected and stain by immunohistochemical stain for K1-10, it is clear the presence K1/10 in green color and the presence of actin (red color) Fig 14 Keratinocyte immunochemical stain for p63 at day 17, red indicates presence of p63, inverse microscope 9400 In the current study we have shown that is possible to obtain keratinocytes from hUCB MSCs in vitro culture, through direct induction We isolated MSCs from hUCB and we cultured up to the 3rd passage and induced them into keratinocytes using a specific medium composed of PCM-SFM MSCs started to change shape after few days of culture in a new medium, assuming the typical round-cuboidal keratinocyte shape Positive immunohistochemical stain 123 132 Fig 17 Immunohistochemistry result with p63 antibody, positive control: 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