Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Suchánek, Jakub; Soukup, Tomáš; Ivančaková, Romana; Karbanová, Jana; Hubková, Věra; Pytlík, Robert; Kučerová, Lenka

doi:10.14712/18059694.2017.82

Acta Medica > Archive > 2007, Vol. 50 > Issue 3 > Human Dental Pulp Stem Cells – Isolation…

Acta Med. 2007, 50: 195-201

https://doi.org/10.14712/18059694.2017.82

Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Jakub Suchánek^a, Tomáš Soukup^b, Romana Ivančaková^a, Jana Karbanová^b, Věra Hubková^a, Robert Pytlík^c, Lenka Kučerová^d

^aCharles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Department of Dentistry, Hradec Králové, Czech Republic
^bCharles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Department of Histology and Embryology, Hradec Králové, Czech Republic
^cCharles University in Prague, 1st Medical Faculty in Prague, Teaching Hospital, 1st Department of Medicine, Prague, Czech Republic
^dCharles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Department of Clinical Genetics, Hradec Králové, Czech Republic

Received March 1, 2007
Accepted August 1, 2007

Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflick’s limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60–90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflick’s limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes.

Keywords

Dental pulp, Stem cells, Isolation, Cultivation, Doubling time, Hayflick’s limit.

Funding

Work was supported by grant project of the Ministry of Health, Czech Republic NR 9182–3/07, by the research project of the Ministry of Education, CR MSM 0021620820 and internal grant for 1st year Ph.D. students, No. 84124 of Charles University in Prague, Faculty of Medicine in Hradec Králové.