In vitro culture of keratinocytes from human umbilical cord blood mesenchymal stem cells the saigonese culture

In vitro culture of Keratinocytes from human umbilical cordblood mesenchymal stem cells: the Saigonese culture Tran Cong Toai•Huynh Duy Thao•Ciro Gargiulo• Nguyen Phuong Thao• Tran Thi T

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: 6 January 2010 / Accepted: 3 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 keratino-cytes 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 7 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
UCB
Keratinocyte culture
Cell culture

Introduction 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 transplanta-tion (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 pathol-ogies such as diabetes, Crohn’s disease, osteogenesis imperfect (OI), rheumatoid arthritis (RA) and osteo-arthritis (OA) (Toai et al 2009; Lee et al 2004;

T C Toai (&)
H D Thao
N P Thao

T T T Thuy
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
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 1 Hospital

in HCMC, Ho Chi Minh City, Vietnam

DOI 10.1007/s10561-010-9174-8

Riordan et al 2007; Kogler and Wernet 2006; Kim

et al.2004; Reddi2007; Koblas et al.2005; De Bari

and Dell’Accio 2007; Tuan and Chen2006; 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

immune-modulatory 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 Laughlin2005; Koc and Lazarus2001; Chao

et al 2004; Majhal et al 2006; Sasaki et al 2008;

Stocum2006)

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

advan-tage 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 1 or 2 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

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 naı¨ve Th0 phenotype that show a restricted response triggered by recipient alloantigens (Tse and Laughlin2005) 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 3 to 7 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 1 9 106at room temperature 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 collected and

seeded in flasks (Nunc, Wiesbaden-Germany)

con-taining 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 2 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 2 ml

solution of Trypsin–EDTA (Gibco, Brl-USA) and

incubated for 5 min at 37°C with 5% CO2. After

5 min, 2 ml of IMDM plus 15% FBS was added,

cells were removed, aspired and transferred in a tube

for centrifugation for 5 min 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

keratino-cytes 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 3

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 7 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

Keratinocyte medium composition Keratinocyte medium is composed of 1 part of PCM and 9 parts of SFM

PCM medium DMEM medium (Gibco Grand Island NY-USA), HEPES 1 M (Sigma Ultra), FBS 15%, EGF 100 mg/

ml (Gibco Invitrogen Corporation), Cholera toxin

10-7M (List biological Laboratories Inc.), Hydrocor-tisone 0,1 mg/ml (Westcort USA), Penicillin/Strep-tomycin 2009

Defined keratinocyte-SFM Medium (Gibco Invit-rogen 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 5 min and washed by alcohol

100, 90, 80%, respectively for 1 min each passage For antigen retrieval, slides were immersed in buffer solution at pH 9 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 min 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 min 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 1 min with 0.1% triton 1009 (ICN Biomedicals; Aurora, OH) in PBS, before staining with the mouse anti-human keratin 1/10 monoclonal

antibody (1ug/ml, CBL266, Chemicon/Millipore,

North Ryde, NSW, Australia) for 4 h, followed by

AlexaFluor488 donkey-anti mouse (1:100, Molecular

Probes/Invitrogen, Mulgrave, Victoria, Australia) for

1 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/Invitro-gen) 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

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 2 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 3 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 2

assuming a more round-cuboidal conformation

typ-ical of keratinocyte like cells (Figs.5,6,7,8,9,10)

Cells were constantly monitored by inverse

micro-scope 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)

Discussion

By definition, MSCs show regular features includinga fibroblast like morphology, a high rate of self-renewal aptitude, an unusual capacity of differenti-ating 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; Reddi2007; Sasaki

et al 2008) Because MSCs, in our experience, qualify by these criteria, we named these cells mesenchymal stem cells in the current article

Fig 1 Human UCB monoclonal primary stem cells after

2 days of culture in IMDM ?10% FBS, inverse microscope

9 100

Fig 2 Primary cells from hUCB after 5 days of culture start assuming fibroblast like shape, 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 kerati-nocyte 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

Fig 3 After 7 days of culture cells assumed a complete

mesenchymal morphology, inverse microscope 9100

Fig 4 MSCs at day 10 start forming a compact and dens

monolayer the confluence is nearly 60–70%, inverse

micro-scope 9100

Fig 5 MSCs from UCB in keratinocyte medium culture after

3 days (2009)

Fig 6 MSCs from UCB in keratinocyte medium culture after

3 days (1009)

process by refilling of cells and extracellular

compo-nents (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)

Fig 7 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

Fig 8 MSCs, control group, 80% confluence, 15 days 9100.

MSCs in keratinocyte medium after 2 weeks culture c.ca It’s

possible to visualize the presence of round shape keratinocyte

cells 9100

Fig 9 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 11 keratinocytes immunohistochemical stain with p63 at day 14, red indicates presence of p63 9200

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 kerat-inocyte shape Positive immunohistochemical stain

Fig 12 Control group stained with Giemsa 9200

Fig 13 Keratinocyte immunochemical stain for p63 at day 14,

red indicates presence of p63 and blue indicates the nucleus,

inverse microscope 9400

Fig 14 Keratinocyte immunochemical stain for p63 at day 17,

red indicates presence of p63, inverse microscope 9400

Fig 15 Keratinocytes stained for keratin 1/10, green color, nuclei blue color, by electronic microscope

Fig 16 Control group stain, electronic microscope Keratino-cytes at 7 day culture were collected and stain by immunohis-tochemical stain for K1-10, it is clear the presence K1/10 in green color and the presence of actin (red color)

for K1-10 and p63 were found in confluent cultures,

the expression of these factors seems to rely on the

presence of keratinocytes in culture In conclusion,

this method presents several advantages it is easy to

perform, there is no need for feeder layers and it can

be accomplished in typical closed culture flasks,

limiting the chances of bacterial contamination In

addition, although this is still at the in vitro stage, the

results confirm two types of data Firstly this

substantiates that MSCs are capable of keratinocyte

differentiation and secondly, it shows that MSCs

from hUCB retain a potential capacity in the skin

regeneration process that is of high value in clinical

applications

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