Dhingra Sanjiv - Adult Stem Cells: Methods and Protocols

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Springer Science+Business Media LLC 2017

Paolo Di Nardo , Sanjiv Dhingra and Dinender K. Singla (eds.) Adult Stem Cells Methods in Molecular Biology 1553 10.1007/978-1-4939-6756-8_1

1. A Simple Protocol to Isolate, Characterize, and Expand Dental Pulp Stem Cells

Federica Di Scipio 1, Andrea Elio Sprio 1, Maria Elisabetta Carere 1, Zhiqian Yang 1 and Giovanni Nicolao Berta 1

(1)

Department of Clinical and Biological Sciences, University of Turin, Regione Gonzole 10, 10043 Orbassano (Turin), Italy

Giovanni Nicolao Berta

Email:

Abstract

Adult stem cells reside in body tissues to preserve organs and whole organism homeostasis. They are acquiring a prominent role in the contemporary medicine. Many protocols to isolate and cultivate adult stem cells have been so far described, though they are often lengthy, laborious, and require very expansive instruments, materials, and reagents. On this basis, we describe a simple, cheap but at the same time functional method to: (1) isolate dental pulp stem cells (DPSC), (2) expand and cultivate DPSC, (3) cryopreserve DPSC, (4) characterize DPSC, and (5) differentiate DPSC into both mesenchymal and non-mesenchymal lineages.

Key words

Neural crest Dental pulp stem cells Cell extraction Characterization Expansion Differentiation

Introduction

Stem cell (SC) research is one of the most interesting and promising areas of contemporary medicine . SC were postulated more than one century ago [], thus paving the way for the current regenerative medicine era. From then, adult SC were retrieved in quite all body tissues , in which they reside to preserve organs and whole organism homeostasis. In the last two decades, in response to the increasing interest and request about artificial tissues, scientists have tried to cultivate resident cells, create engineered tissues, and design treatment for virtually every organ of the human body. In this Gold Rush, a multitude of adult stem cells have been isolated, characterized using a plethora of different and frequently generic stemness markers, and induced to differentiate toward several lineages. Nevertheless, the described features were frequently contradictory, depending on the anatomical site from which SC were extracted as well as on methods and equipment employed.

In this scenario, the dental pulp could represent an important SC source. In fact, it is an ontogenetically derivative of the only interesting thing about vertebrates [].

In particular, during the odontogenesis, dental pulp derives from an ectomesenchymalization process in which the first pharyngeal arch epithelium interacts with the neural crest as mesenchymal counterpart. Moreover, these cells are early compartmentalized within the dental follicle and thus isolated and protected from massive differentiation stimuli that drive the jawbone development in the adjacent tissues (Fig. ].

Fig. 1

Tricromical stained picture of a rat head at the 14 days of intrauterine life. Note the differences between the already differentiated Meckels cartilage (#), from which the lower jawbone derives, and the dental papilla (*), entrapped between the enamel organ () and a fibrous connective membrane (^). In blue , mineralization confirms the status of differentiated tissues

Based on these evidences, it does not surprise that despite closely sharing the antigenic phenotype of bone marrow stromal cells, the dental pulp stem cells (DPSC) have exhibited a higher proliferation rate and demonstrated to differentiate in-vitro into bone, cartilage, adipose, neuronal, and cardiac precursors []. Nevertheless, they are so far underestimated to a certain extent, being up to now predominantly evaluated and employed for in vivo dental /calcifying tissue recovery.

Although in some mammals (e.g., rodents) incisive teeth grow throughout life inasmuch as open-rooted [].

Finally, despite consisting of a small amount of tissue, if compared to other stem cell sources, dental pulp retains the dual advantage of a richer content in stem cells and a less harmful/invasive procedure for their collection []. Here, we provide a simple, cheap, and reliable method to extract, isolate, and propagate adult stem cells from the dental pulp of both humans and animal models, without the employment of cell feeder or expensive medium supplements.

Materials

1. Phosphate buffered saline solution, pH 7.4.
2. Dulbeccos Phosphate Buffered Saline Modified, without calcium chloride and magnesium chloride.
3. Penicillin G (200 U/mL), Gentamicin sulphate (80 mg/mL) and 5 mg/mL Amphotericin B.
4. Collagenase/Dispase solution: 3 mg/mL collagenase type I and 4 mg/mL dispase II.
5. RPMI-1640 medium.
6. Fetal calf serum.
7. Trypsin/EDTA solution.
8. Dimethyl sulfoxide.
9. Paraformaldehyde.
10. Toluidine Blue solution .
11. Trypan Blue solution.
12. TRI Reagent solution (Sigma-Aldrich, Saint Louis, USA).
13. Chloroform.
14. Isopropyl alcohol.
15. Ethanol absolute, 99.8 %.
16. Deoxyribonuclease I kit (Fermentas International, Inc., Burlington, Canada).
17. RevertAid RT Reverse Transcription Kit (Fermentas International).
18. 10 PCR Buffer II (Roche Applied Science, Indianapolis, USA).
19. 25 mM MgCl2 (Fermentas International).
20. 10 mM dNTP (Fermentas International).
21. Specific 10 mM primer (Sigma-Genosys).
22. Taq DNA Polymerase, recombinant (Fermentas International).
23. Triton X-100.
24. Fluoroshield Mounting Medium With DAPI (Abcam, Cambridge, UK).
25. Bovine serum albumin.
26. Sodium azide (NaN3).
27. Facs buffer, PBS, 0.51 % BSA or 510 % FCS, 0.1 % NaN3.
28. Adipogenic inductive medium; RPMI 1640 supplemented with 10 % (v/v) FCS, 1.7 mM insulin, 1 mM dexamethasone, and 0.5 mM methylisobutylxanthine, 100 U/mL penicillin G, 40 mg/mL gentamicin sulfate, and 2.5 mg/mL amphotericin B (Sigma-Aldrich).
29. Nile red staining solution.
30. Fluorescence Mounting Medium.
31. Osteogenic inductive medium; RPMI 1640 supplemented with 10 % (v/v) FCS, 10 mM b-glycerophosphate, 0.05 mM ascorbic acid, and 100 nM dexamethasone, 100 U/mL penicillin G, 40 mg/mL gentamicin sulfate, and 2.5 mg/mL amphotericin B (Sigma-Aldrich).
32. Alkaline phosphatase staining kit (BioOptica Milano SpA, Milano, Italy).
33. Chondrogenic inductive medium ; RPMI 1640 supplemented with 10 % (v/v) FCS, 10 ng/mL TGF-3, 0.025 mM ascorbic acid, and 100 nM dexamethasone, 100 U/mL penicillin G, 40 mg/mL gentamicin sulfate, and 2.5 mg/mL amphotericin B (Sigma-Aldrich).
34. -Mercaptoethanol.
35. Retinoic acid.
36. Neuroglial inductive medium; RPMI 1640 supplemented with 10 % (v/v) FCS and 5 ng/mL platelet-derived growth factor, 10 ng/mL basic fibroblast growth factor, 252 ng/mL glial growth factor, and 14 mM forskolin (Sigma-Aldrich).
37. Antiboby against Neuron growth-associated protein 43 (GAP43) (Santa Cruz Biotechnology, Santa Cruz, CA).
38. Antiboby against Glial fibrillary acidic protein (GFAP) (DakoCytomation, Dako Italia SpA, Milano, Italy).

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