高雄醫學大學口腔醫學院 研究日 第五屆學術研討會 26 april, 2013 human dental...

高雄醫學大學口腔醫學院 高雄醫學大學口腔醫學院 研究日研究日 第五屆學術研討會第五屆學術研討會 26 April, 2013

26 April, 2013

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate

hepatic-like differentiationYuk-Kwan Chen1,3 Anderson Hsien-Cheng Huang1 Tien-Yu Shieh2,4 Li-Min Lin1,3

Departments of 1Oral Pathology, 2Oral & Maxillofacial Surgery, Faculty of Dentistry, College of Dental Medicine, Kaohsiung Medical University; Division of Dentistry, 3Departments of Oral Pathology, 4Oral & Maxillofacial Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan

Overall Introduction

• Adult stem cells (ASCs) have been isolated from various tissues including bone marrow, dental, liver, blood vessel, adipose, and neuronal tissues (Barrilleaux et al., 2007)

• Stem cells isolated from dental epithelium and dental pulp are capable of differentiating to a dentin-pulp-like complex, which is similar to that of normal human teeth (Gronthos et al., 2000; Gronthos et al., 2002)

• Human dental pulp stem cells (hDPSCs) share similar gene expression profiles and differentiation capacity to that of bone marrow mesenchymal stem cells (BMSCs) (Shi et al., 2001)

• hDPSCs are also capable of differentiating to osteogenic, adipogenic, chondrogenic, and neurogenic lineages (Gronthos et al., 2000; Gronthos et al., 2002; Iohara et

al., 2006), but not for hepatic differentiation of cryopreserved dental pulp tissues of vital extracted teeth with disease

Overall Introduction

• Part (1) Different Cryopreservations of Human Dental Pulp Tissues from Vital Extracted

Teeth with Disease

• Part (2) Hepatic-Like Differentiation from Cryopreserved Dental Pulp Tissues of Vital Extracted Teeth with Disease

The current study contains the following

two parts of experiments

< Part 1 Study >Different Cryopreservations of

Human Dental Pulp Tissues from Diseased Teeth

Group A Freshly derived dental pulp tissues, n = 20

Age: range - 6-74 y/o average - 26.5 y/oSex: M/F: 8/12

Group BLiquid nitrogen (N2) stored dental pulp tissues, n = 20

Age: range - 6-49 y/oaverage - 23.4 y/o

Sex: M/F: 5/15

Group C Liquid nitrogen (N2) stored intact teeth, n =10

Age : range - 8-52y/o average - 27.7y/o Sex: M/F: 5/5

Total sample size, n = 50

Age: range - 6-74 y/o average - 25.5 y/oSex: M/F: 18/32

M/F: Male/Female

A cohort of 50 vital extracted teeth with diseases (residual root, supernumerary tooth, periodontitis, pericoronitis, and tooth fracture) were randomly divided as:

Group A (n = 20) : Freshly derived dental pulp tissues

Group B (n = 20) : Liquid nitrogen stored dental pulp tissues for 30 days (dump-freezing process at -1°C/min for over 8-h in -80°C after immersing in 1ml culture medium with 10% DMSO for 2h at 4°C)

Group C (n = 10) : Liquid nitrogen stored intact teeth for 30 days (dump-freezing process at -1°C/min for over 8-h in -80°C after immersing in 1ml culture medium with 10% DMSO for 2h at 4°C)

Materials

Sex distribution of the total sample size (n = 50)

Distribution of sex

02468

10121416

Fresh (N=20) Liq. N2 cryopresevationpulp(N=20)

Liq. N2 cryopresevationintact tooth(N=10)

numb

ers of

samp

les

Male

Female

Num

ber

of s

ampl

es

Age distribution of the total sample size (n = 50)

Distribution of Age

0

2

4

6

8

10

12

0~19 20~39 40~59 60 or over

num

bers

of s

ampl

es

Fresh(N=20)

Liq. N2cryopresevationpulp(N=20)

Liq. N2cryopresevationintacttooth(N=10)

Nu

mb

er

of s

am

ple

s

Tooth positions distribution of the total sample size (n = 50)

Distribution of tooth samples

0

1

2

3

4

5

6

7

8

Decidous anterior Decidous posterior Permanent anterior Permanentpremolar

Permanent molar supernumerary

Tooth position

num

bers

of s

ampl

es

Fresh(N=20)

Liq. N2 cryopresevationpulp(N=20)

Liq. N2 cryopresevationintact tooth(N=10)

Num

ber

of

sam

ples

DeciduousAnterior

DeciduousPosterior

Growth rate analyses by MANOVA on sex, age & tooth position categories for Group A samples

The whole model result of MANOVA on Group A (n = 20)

TestValue Approx. F NumDF DenDF Prob>F

Wilks' Lambda 0.0979072 0.6674 36 27.97 0.8741

The whole model result of MANOVA on Group B samples (n = 20)

TestValue Approx. F NumDF DenDF Prob>F

Wilks' Lambda 0.1887049 0.7114 28 33.872 0.8203

Growth rate analyses by MANOVA on sex, age & tooth position categories for Group B samples

A circumference groove of 0.5-1.0 mm in depth was cut around the entire tooth (arrows) using an aseptic high speed handpiece bur after cleaning the tooth surface with DPBS (A). The tooth was split using a chisel (B), dental pulp was then exposed (C) and the underlying pulp tissue was extracted using an endodontic file placing in a container with DPBS for subsequent procedures of culture (D)

Cryopreservation vial containing the intact tooth

Group AGroup B

Group C

Isolation and culture of human dental pulp stem cells (DPSCs )

Colony forming unit (CFU)

Group A samples (successful isolation, n = 20) A homogenous population predominantly long and spindle shaped cells (7-day culture, 100). Colony forming units of hDPSCs from naked eye view with crystal violet staining (A), (B, 40)

A B

Methods and Results

Group B samples (successful isolation, n = 20) A homogenous population predominantly long and spindle shaped cells (7-day culture, 100). Colony forming units of hDPSCs from naked eye view with crystal violet staining (A), (B, 40)

A

B

Group C samples (n = 10; successful isolation, n = 2) A homogenous population predominantly long and spindle shaped cells(7-day culture, 100). Colony forming units of hDPSCs from naked eye view with crystal violet staining (A), (B, 40)

A B

Growth rate analysesThe hDPSCs of Groups A–C had a high proliferation rate during the early passages, but decreased gradually in culture.

Osteogenic, adipogenic, and chondrogenic differentiation

Adipogenic differentiation with oil-Red-O stain (400)

A B C

Group A samples (successful isolation, n = 20)

OsteogenicOsteogenic

Chondrogenic differentiation with alcian blue stain (100)

Osteogenic differentiation with alizarin Red S stain (40)

AdipogenicAdipogenic ChondrogenicChondrogenic

Group B samples (successful isolation, n = 20)

Osteogenic differentiation with alizarin Red S stain (100)

A B

C

OsteogenicOsteogenic

Chondrogenic differentiation with alcian blue stain (40)

Adipogenic differentiation with oil-Red-O stain (400)

AdipogenicAdipogenic ChondrogenicChondrogenic

Group C samples (successful isolation, n = 2)

Adipogenic differentiation with Oil-Red-O stain (200)

A B C

OsteogenicOsteogenic

Chondrogenic differentiation with alcian blue stain (100)

Osteogenic differentiation with Alizarin Red S stain (400)

AdipogenicAdipogenic ChondrogenicChondrogenic

Reverse transcription-polymerase chain reaction (RT-PCR)

GAPDH (596-bp) Osteonectin (323-bp)Nestin (810-bp) Nanog (389-bp)Rex-1 (470-bp) Oct-4 (205-bp)

RT-PCR for stem cell (Oct-4, Nanog & Rex-1) and differentiation (Osteonectin, Nestin and PPARγ) markers of Group A samples (successful isolation, n = 20)

GAPDH (596-bp)PPARγ (391-bp)

Marker H2O

RT-PCR for stem cell (Oct-4, Nanog and Rex-1) and differentiation (Osteonectin, Nestin and PPARγ) markers of Group B samples (successful isolation, n = 20)

GAPDH (596-bp) Osteonectin (323-bp)Nestin (810-bp) Nanog (389-bp)Rex-1 (470-bp) Oct-4 (205-bp)

GAPDH (596-bp)PPARγ (391-bp)

Marker H2O

RT-PCR for stem cell (Oct-4, Nanog & Rex-1) and differentiation (Osteonectin, Nestin and PPARγ) markers of Group C samples (successful isolation, n = 2)

GAPDH (596-bp) Osteonectin (323-bp)Nestin (810-bp) Nanog (389-bp)Rex 1 (470-bp) Oct4 (205-bp)

GAPDH (596-bp)PPARγ (391-bp)

Marker H2O

Flow cytometry

CD29, CD34, CD45, CD90, CD105 are important markers for human bone marrow stem cells which show similar expression to our isolated hPDSCs (Black line: markers of isotype; Red line: markers of interest)

Flow cytometry of the isolated hDPSCs of Group A (successful isolation, n = 20)

Flow cytometry of the isolated hDPSCs of Group B samples (successful isolation, n = 20)

CD29, CD34, CD45, CD90, CD105 are important markers for human bone marrow stem cells which show similar expression to our isolated hPDSCs (Black line: markers of isotype; Red line: markers of interest)

Flow cytometry of the isolated hDPSCs of Group C samples (successful isolation, n = 2)

CD29, CD34, CD45, CD90, CD105 are important markers for human bone marrow stem cells which show similar expression to our isolated hPDSCs (Black line: markers of isotype; Red line: markers of interest)

Western blot analyses of the isolated hDPSCs of Group A revealed stem-cell markers (Rex-1 and Oct-4) and differentiation marker (Nestin)

Relative expression level was measured and normalized to expression level of the positive control

Relative expression level was measured and normalized to expression level of the positive control

Western blot analyses of the isolated hDPSCs of Groups B & C revealed stem-cell markers (Rex-1 and Oct-4) and differentiation marker (Nestin)

Relative expression level was measured and normalized to expression level of the positive control

Relative expression level was measured and normalized to expression level of the positive control

Discussion (1)

• Previous researches on hDPSCs have only focused on the use of dental pulp tissues from healthy deciduous incisors and permanent third molars (Sloan & Smith 2007)

• The isolation of hDPSCs from a cohort of vital extracted teeth with diseases encompassing periodontitis, supernumerary teeth, inflamed malpositioned teeth, root resorption and pericoronitis, as well as fractured teeth, as successfully demonstrated in the current study further expands the potential sources of hDPSCs

Discussion (2)

• Although there have been low rates of successful isolation of hDPSCs from intact teeth after cryopreservation, whole teeth have been successfully cryopreserved and thawed for the purpose of reimplantation (Schwartz & Rank, 1986; Paulsen et al., 2006)

• Temmerman et al. (2010) proved that cryopreservation of human pulp tissue would be possible if the cryoprotective agent could reach the entire pulp. Consequently, if a novel cryopreservation modality could be invented, as demonstrated by Lee et al. (2010), improvements in cryopreservation efficiency could be attained

< Part 2 Study>Hepatic-Like Differentiation from

Cryopreserved Dental Pulp Tissues of Vital Extracted Teeth with Disease

Materials • The 3rd-passage hDPSCs obtained from the extracted pulp tissues of Groups A (freshly-derived dental pulp tissues) and B (liquid nitrogen-stored dental pulp tissues) were used for hepatic differentiation

• Negative controls for hepatic differentiationEach of the samples of the 3rd-passage hDPSCs derived from Groups A and B respectively was treated with basal medium with penicillin/streptomycin (Invitrogen) and glutamine (Invitrogen) but without any growth factor or supplements, and these treated samples were used as hepatic differentiation negative controls.

• Negative controls for hepatic differentiationEach of the samples of the 3rd-passage hDPSCs derived from Groups A and B respectively was treated with basal medium with penicillin/streptomycin (Invitrogen) and glutamine (Invitrogen) but without any growth factor or supplements, and these treated samples were used as hepatic differentiation negative controls.

• Positive control for hepatic differentiationA well-differentiated hepatocellular carcinoma (HCC) cell line (BCRC: 60169, Food Industry Research and Development Institute, Taiwan) was commercially available and employed as the positive control for hepatic differentiation

• Positive control for hepatic differentiationA well-differentiated hepatocellular carcinoma (HCC) cell line (BCRC: 60169, Food Industry Research and Development Institute, Taiwan) was commercially available and employed as the positive control for hepatic differentiation

(A) Morphological change of hDPSCs upon hepatic differentiation was noted 14 days after the cell cultures were exposed to differentiation media and still be observed up to 42 days (200) for Groups A & B

(A) Morphological change of hDPSCs upon hepatic differentiation was noted 14 days after the cell cultures were exposed to differentiation media and still be observed up to 42 days (200) for Groups A & B

Methods and Results

(B) Reverse transcription-polymerase chain reaction of various hepatic markers after various durations of differentiation of hDPSCs for Groups A & B

(B) Reverse transcription-polymerase chain reaction of various hepatic markers after various durations of differentiation of hDPSCs for Groups A & B

M: Molecular weight markerN: H2OLane 1: 1-dayLane 2: 7-dayLane 3: 14-dayLane 4: 28-dayLane 5: 42-day

M: Molecular weight markerN: H2OLane 1: 1-dayLane 2: 7-dayLane 3: 14-dayLane 4: 28-dayLane 5: 42-day

Relative expression level of various hepatic markers was measured and normalized to GAPDH mRNA level

Day 1 Day 7 Day 28 Day 42

C

(C) Periodic acid-Schiff staining for glycogen –Hepatic differentiated hDPSCs revealed positive staining starting from the 7th day of differentiation-medium culturing and still be observed up to 42 days (100) for Groups A & B

(C) Periodic acid-Schiff staining for glycogen –Hepatic differentiated hDPSCs revealed positive staining starting from the 7th day of differentiation-medium culturing and still be observed up to 42 days (100) for Groups A & B

Day 14Day 1 Day 28 Day 42

D

(D) Immunofluorescence staining for low-density lipoprotein –Hepatic differentiated hDPSCs revealed positive staining starting from the 14th day of differentiation-medium culturing andstill be observed up to 42 days (200) for Groups A & B

(D) Immunofluorescence staining for low-density lipoprotein –Hepatic differentiated hDPSCs revealed positive staining starting from the 14th day of differentiation-medium culturing andstill be observed up to 42 days (200) for Groups A & B

E

Day 1

(E) Immunofluorescence staining for albumin –Hepatic differentiated hDPSCs revealed positive staining starting from the 14th day of differentiation-medium culturing andstill be observed up to 42 days (200) for Groups A & B

(E) Immunofluorescence staining for albumin –Hepatic differentiated hDPSCs revealed positive staining starting from the 14th day of differentiation-medium culturing andstill be observed up to 42 days (200) for Groups A & B

(F) Mean urea concentrations of all the cryopreserved samples in the different weeks of differentiation (blue line): Urea production by hepatic differentiated hDPSCs was noted starting from the 6th week of differentiation-medium culturing and increased as observed up to the 8th week; HCC cell line also demonstrated positive findings for urea assay (red line)

(G) Western blot revealed hepatocyte nuclear factor (HNF) 4 expression in both sample and control (HCC cell line)

(H) Genetic analysis revealed 100% normal karyotype

Hepatic differentiations of negative controls and positive control

(A1) Morphology of undifferentiated hDPSCs (negative control) remained unchanged as observed up to 42 days (100)(A2) A well-differentiated hepatocellular carcinoma (HCC) cell line used as the positive control for hepatic differentiation (200)

(B1) Undifferentiated hDPSCs demonstrated negative findings upon reverse transcription-polymerase chain reaction of various hepatic markers as observed up to 42 days

(B1) Undifferentiated hDPSCs demonstrated negative findings upon reverse transcription-polymerase chain reaction of various hepatic markers as observed up to 42 days

M: Molecular weight markerLane 1: GAPDH (596-bp)Lane 2: H2OLane 3: alpha-fetoproteinLane 4: albuminLane 5: cytokeratin-18Lane 6: tryptophan 2,3-dioxygenase Lane 7: glucose-6-hosphataseLane 8: CCAAT/ enhancer-binding proteins αLane 9: hepatocyte nuclear factor-1α Lane 10: cytochrome P450 family-1 subfamily-A, polypeptide-1

M: Molecular weight markerLane 1: GAPDH (596-bp)Lane 2: H2OLane 3: alpha-fetoproteinLane 4: albuminLane 5: cytokeratin-18Lane 6: tryptophan 2,3-dioxygenase Lane 7: glucose-6-hosphataseLane 8: CCAAT/ enhancer-binding proteins αLane 9: hepatocyte nuclear factor-1α Lane 10: cytochrome P450 family-1 subfamily-A, polypeptide-1

(B2) Reverse transcription-polymerase chain reaction for various hepatic markers were positive for the HCC cell line

(C1) Periodic acid-Schiff staining for glycogen – Undifferentiated hDPSCs demonstrated negative findings when observed up to 42 days (100)(C2) Periodic acid-Schiff staining for glycogen was positive for the HCC cell line ( 100)

(D1) Immunofluorescence staining for low-density lipoprotein –Undifferentiated hDPSCs demonstrated negative findings when observed up to 42 days (200)(D2) Immunofluorescence staining of low-density lipoprotein uptake was positive for the HCC cell line (200)

(E1) Immunofluorescence staining for albumin – Undifferentiated hDPSCs demonstrated negative findings observed up to 42 days (200)(E2) Immunofluorescence staining of albumin was positive for the HCC line (200)

(F) Undifferentiated hDPSCs demonstrated negative findings for urea assay when observed up to the 8th week

(F) Undifferentiated hDPSCs demonstrated negative findings for urea assay when observed up to the 8th week

• After hepatic differentiation, markers of FP, Alb, C/EBPα, HNF1α and especially, CYP1A1 (a key enzyme of xenobiotic effect of hepatic cells) as well as HNF4α (a key hepatic developmental associated transcription factor for MSC-to-hepatic-like cell conversion) were found to be expressed in the cell cultures derived from the present study cohort indicating that, as reported for MSCs isolated from human bone marrow and umbilical cord blood (Tang et al., 2006), the dental pulp tissues of vital extracted teeth with disease have the potential to differentiate into hepatic-like cells even after cryopreservation

Discussion (1)

• Positive urea production and positive glycogen storage in hDPSC-derived hepatic-like cells obtained from cryopreserved dental pulp tissues of vital extracted teeth with disease are demonstrated in the current study

• We observed a significantly increase in urea production as compared between 6 and 7 weeks of differentiation but a non-significantly increase between 7 and 8 weeks of differentiation, perhaps indicating that the hepatic differentiation of the cryopreserved DPSCs have nearly approached a peak value upon 8 weeks of differentiation

Discussion (2)

• Urea concentrations of the current study were observed to be compatible to the values expressed by the induced pluripotent stem cells (iPS)-derived hepatic cells in the recent study of Ghodzizadeh et al. (2010)

• In summary, the differentiated cells of the present study appear to be functionally close to normal hepatic-like cells

Discussion (3)

• With normal karyotyping of the differentiated cells of the present study as well as using appropriate hepatic differentiation positive and negative controls, the current study demonstrates normal karyotype hepatic-like cells are derived from cryopreserved dental pulp tissues of extracted vital teeth with disease

• This study provides evidences that hDPSC from cryopreserved dental pulp tissues of vital extracted teeth with disease could be one of the potential candidates of hepatic-like cells

Discussion (4)

• To conclusively demonstrate the potential application of hDPSC-derived hepatic-like cells, one should be dealt with the drawback that the cell proliferation has been declined with passage

• hDPSCs constitute a minor population of total dental pulp cells and, depending on the size of the collected dental pulp tissues, a range of 6 to 20 cells were able to be isolated from one dental pulp tissue in the current study. Hence, cell quantity would be a key issue in spite of an excellent proliferating capacity

Discussion (5)

• Overall, the results obtained from this study allowed us to conclude that hDPSCs not only can give rise to hepatic-like cells from the isolated dental pulp tissues of vital extracted teeth with disease (even after cryopreservation of the dental pulp tissues followed by digestion and culturing post-thawing) but also the differentiated cells possess normal karyotype and are functionally close to normal hepatic-like cells

Overall Conclusion

• We believe that, if in future, the issue of hDPSCs being a minor population of total dental pulp cells can be manipulated and an enough amount of functional hepatic-like cells could be obtained from the dental pulp tissues, then, hDPSCs derived from cryopreserved dental pulp tissues of vital extracted teeth with disease to be used as a possible source for autogenic transplantation for tissue engineering-based approaches for treatment of hepatic conditions would not be impossible

Overall Conclusion

Professor Shieh TY Professor Lin LM

Associate Professor Chen YK

Assistant ProfessorHuang AHC

Kaohsiung Medical UniversityDepartment of Oral Pathology

Acknowledgment

Thank you very much

Glory to the Lord