[methods in molecular biology] somatic stem cells volume 879 || identification, isolation,...

443

Shree Ram Singh (ed.), Somatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 879,DOI 10.1007/978-1-61779-815-3_26, © Springer Science+Business Media, LLC 2012

Chapter 26

Identi fi cation, Isolation, Characterization, and Banking of Human Dental Pulp Stem Cells

Virginia Tirino , Francesca Paino, Alfredo De Rosa, and Gianpaolo Papaccio

Abstract

Dental pulp stem cells (DPSCs) can be found within the “cell rich zone” of the dental pulp. Their embryonic origin, from neural crests, explains their multipotency. Up to now, it has been demonstrated that these cells are capable of producing bone tissue, both in vitro and in vivo, as well as a simil-dentin tissue, in vitro. In addition, it has been reported that these cells differentiate into adipocytes, endotheliocytes, melanocytes, neurons, and glial cells and can be easily cryopreserved and stored for long periods of time and retain their multipotency and bone-producing capacity. Moreover, recent attention has been focused on tissue engineer-ing and on the properties of these cells: several scaffolds have been used to promote 3D tissue formation and studies have demonstrated that DPSCs show good adherence and bone tissue formation on microconcavity surface textures. In addition, adult bone tissue with good vascularization has been obtained in grafts. Interestingly, they seem to possess immunoprivileges as they can be grafted into allogenic tissues and seem to exert anti-in fl ammatory abilities, like many other mesenchymal stem cells. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients.

Therefore, their isolation, selection, differentiation, and banking are of great importance. The isola-tion technique used in most laboratories is based on the use of fl ow cytometry with cell sorter termed FACS ( fl uorescent activated cell sorter). It is now important to obtain new methods/protocols to select and isolate stem cells without staining by fl uorescent markers or use of magnetic beads.

These new procedures should be based on biophysical differences among the different cell populations in order to obtain interesting peculiarities for implementation in biomedical/clinical laboratories. It is empha-sized that the new methods must address simplicity and short times of preparation and use of samples, com-plete sterility of cells, the potential disposable, low cost and complete maintenance of the viability, and integrity of the cells with real-time response for subsequent applications in the biomedical/clinical/surgical fi elds.

Key words: DPSCs , Stem cell identi fi cation , Methods , Strategies , Differentiation , Regeneration

Stem cells are cells delegated to maintain the structural and func-tional integrity of tissues, through the replacement of mature damage cells. Multipotent adult stem cells, as mesenchymal stem cells (MSC),

1. Introduction

444 V. Tirino et al.

are present in all mature tissues of the human body and are thought to reside in a speci fi c area of each tissue where they may remain quiescent (nondividing) for many years until they are activated by disease or tissue injury. A stem cell is self-renewable and capable of differentiating into at least two distinctive cell types ( 1 ) . These two properties must both be satis fi ed for a cell to be de fi ned as a stem cell. Self-renewal denotes that undifferentiated daughter cells are a precise replica and can further replicate many generations without losing their original characteristics ( 2, 3 ) . Cells of an immortalized cell line can replicate many generations, but are generally incapable of multilineage differentiation. Thus, cell lines are not stem cells. Multilineage differentiation refers to the capacity of a single popu-lation of stem cells to differentiate into at least two distinctively different cell types. For example, a single population of MSCs can differentiate into both osteoblasts and chondrocytes. Preosteoblasts can differentiate into osteoblasts, but are incapable of differentiat-ing into other mesenchymal lineages, such as chondrocytes or adi-pocytes, at least not without undergoing dedifferentiation back toward MSCs. In the adult, MSCs maintain physiologically neces-sary tissue turnover and, upon injury or disease, differentiate to launch tissue regeneration. MSCs have been experimentally differentiated into all mesenchymal or connective tissue lineages ( 4, 5 ) and, in many cases, have been used to tissue engineering. Regeneration of tissue structures from stem cells is an insurmount-able effort until advances from several seemingly unrelated disciplines—such as cell and molecular biology, polymer chemistry, molecular genetics, materials science, and mechanical engineering—converged into the self-assembling fi eld of tissue engineering ( 6, 7 ) . To engineer a functional biological structure, cells must be instructed to differentiate and receive positional cues and to syn-thesize the appropriate extracellular matrix molecules in the overall shape and dimensions of the diseased or missing tissues/organs. Biomimetic scaffolds are frequently needed to enable cell growth and differentiation to occur in an environment that has been previ-ously unfamiliar to either biologists or engineers. Large-scale tissue engineering research began to take place in the early 1990s and has grown exponentially ever since. Numerous niches of stem cells within the human body have been studied up to now and include as main sources for tissue repair/regeneration: Dental stem cells, adipose tissue, mesenchymal and connective (MSCs), muscle stem cells (MyoSC), cartilage stem cells, bone-marrow MSCs forming bone. For example, DPSCs have been engineered for bone tissue building and repair. In fact, these cells easily differentiate and pro-liferate into osteoblasts producing already in vitro a woven bone which then, after transplantation into immunosuppressed rats, give rise to a complete, vascularized lamellar bone ( 8– 10 ) . Those fi ndings are of high interest in bone tissue repair due to their ori-gin, their differentiative abilities, proliferation rate, and log lifespan

44526 Identifi cation, Isolation, Characterization, and Banking of Human Dental…

( 11 ) . In a broader overview, the adult stem cells are an excellent resource in a tissue engineering and regenerative medicine as they can be used as an effective therapeutic strategy in demolition pathologies. Regarding the embryonic origin of dental pulp, embryonic cells migrate from the neural crests to reinforce head and neck mesenchyme strongly determining the development of this area of the human body. During the sixth week of embryogen-esis, ectoderm covering the stomodeum begins to proliferate, giv-ing rise to the dental laminae. Reciprocal interactions between ectoderm and mesoderm layers lead to placode formation. One of these thick, ovoid ectodermal structures develops into tooth germs, where cells, belonging to the neural crest, will differentiate into the dental germ, containing both dental papilla and follicle. Therefore, dental pulp is made of ecto-mesenchymal components, containing neural crest-derived cells, which display plasticity and multipoten-tial capabilities ( 12 ) .

Pulp is externally separated from dentin by odontoblasts and by Höhl’s subodontoblastic cells that are preodontoblasts ( 13 ) . Adjacent to this layer, the pulp is rich in collagen fi bers and poor in cells. Then, another, more internal layer, contains progenitor cells and undifferentiated cells, some of which are considered stem cells ( 14 ) . From this layer, undifferentiated cells migrate to various districts where they can differentiate under different stimuli and make new differentiated cells and tissues. The fi nal, innermost layer is the core of the pulp; this area is comprised of the vascular plexus and nerves. Up to the more recent discoveries ( 10, 15 ) , researchers hypothesized that DPSCs were present in this layer ( 16 ) . Actually, only undifferentiated perivascular cells can be found in it.

The third molar tooth germ begins development around the sixth year of life. Until this time, embryonic tissues of dental lamina remain quiescent and undifferentiated within the jaw of the child. Although crown mineralization begins during the eighth year of life, often third molar roots are still incomplete at the age of 18. This means that the structure of those teeth is still immature at this age and a conspicuous pool of undifferentiated cells, resident within the “cell rich zone” of the dental germ pulp, is needed for development.

1. Chlorexidin gel (Forhans). Store at room temperature until exposure date.

2. I Type collagenase (GIBCO, Invitrogen). Store at 4°C until exposure date (see Note 1 ).

3. Dispase (GIBCO, Invitrogen). Store at 4°C until exposure date (see Note 1 ).

2. Materials

2.1. Cell Culture Components


4. Gentamicin solution (Sigma). Store at room temperature until exposure date.

5. PBS w/o Ca 2+ , Mg 2+ (Lonza). Store at 4°C until exposure date.

6. 70- μ m Falcon strainers (Becton & Dickinson). Store at room temperature.

7. MegaCell culture medium (Sigma). Store at 4°C until expo-sure date.

8. Fetal bovine serum, South American Origin (GIBCO, Invitrogen). Store at −20°C until exposure date (see Note 2 ).

9. L -ascorbic acid (Sigma). Store at room temperature until expo-sure date.

10. L -glutamine (Lonza). Store at −20°C until exposure date (see Note 3 ).

11. Penicillin and streptomycin, Pen/Strep (Lonza). Store at −20°C until exposure date (see Note 3 ).

12. α -MEM culture medium (Invitrogen). Store at 4°C until exposure date.

13. EDTA (Sigma). Store at room temperature until exposure date.

14. Bovine serum albumin (BSA) fraction V (Sigma). Store at room temperature until exposure date.

15. Trypsin-EDTA solution (Lonza), stored at 4°C until exposure date.

16. Dimethyl sulfoxide, DMSO (Sigma). Store at room tempera-ture at dark, until exposure date.

1. TGF- β 1 (Abcam). Store at −20°C until exposure date. 2. Neurobasal A medium (1×) (Invitrogen). Store at 4°C until

exposure date and to use within 1 month after its opening. 3. B27 (Invitrogen), stored at 4°C until exposure date. 4. Basic fi broblast growth factor, bFGF (Sigma). Stored at −20°C

until exposure date (see Note 4 ). 5. Epidermal growth factor, EGF (Sigma). Store at −20°C until

exposure date (see Note 4 ). 6. Dexamethasone (Sigma). Store at +4°C until exposure date. 7. Recombinant human insulin (Sigma). Store at +4°C until

exposure date. 8. Indomethacin (Sigma), stored at room temperature until expo-

sure date. 9. 3-Isobutyl-1-methyl-xantina, IBMX (Sigma), stored at −20°C

until exposure date.

2.2. Differentiation Media Components


1. Oil red-O (Sigma). Store at room temperature until exposure date.

2. Toluidine blue (Sigma). Store at room temperature. 3. Paraformaldehyde (Sigma). Store at room temperature (see

Note 5 ). 4. BMPurple solution (Roche). Store at room temperature until

exposure date. 5. Calcein-AM (Dojindo Molecular Technologies). Store at

−20°C until exposure date. 6. Glutaraldehyde (EM grade) (Sigma). Store at room tempera-

ture, until exposure date. 7. Alkaline phosphatase, ALP (Sigma), stored at room tempera-

ture, until exposure date. 8. Osmium tetroxide, OsO 4 (Sigma). Store at room

temperature. 9. Triton X100 (Sigma). Store at room temperature. 10. Fix&Perm Kit (Invitrogen). Store at room temperature, until

exposure date. 11. Epoxy Resins Epon 812 (Sigma). Store at room temperature.

1. CD31 (PECAM-1) (Becton & Dickinson). 2. CD34 PE (Milteniy Biotec). 3. CD44 (MBL, Woburn). 4. CD45 CY (Becton & Dickinson). 5. CD54 (MBL, Woburn). 6. CD90 FITC (Becton & Dickinson). 7. CD117 FITC (c-kit) (Santa Cruz). 8. CD133 PE (Milteniy Biotec). 9. Flk-1 (Santa Cruz). 10. STRO-1 (Dr. Torok-Storb through DBA, Segrate, Milan, Italy). 11. Osteocalcin (Santa Cruz). 12. RUNX-2 (Santa Cruz). 13. Von-Willebrand (domain 1 and 2) (Santa Cruz). 14. Angiotensin-converting-enzyme, ACE (Santa Cruz) (see Note 6 ).

1. Iodide propide solution (Sigma). Store at 4°C in the dark until exposure date.

2. Sodium citrate (Sigma). Store at room temperature until exposure.

3. RNAse A (Sigma). Store at −20°C in the dark until exposure date.

2.3. Staining Components

2.4. Antigens and Conjugates

2.5. Cell Cycle Analyses


1. BD FACSAria II near UV Upgrade kit for 3 laser system (Becton Dickinson).

2. Electron microscope (Philips 400 S). 3. Fluorescence microscope (Nikon TE 2000-S).

The stem population, isolated and cultured in vitro, includes a mosaicism of different cell types with different stages of differentia-tion. Numerous laboratories use the same techniques of detection and isolation of stem cells (SC): tissue surgical removal under sterile conditions, digestion in collagenase/dispase, detection and selection by selective markers. For example, SC isolated from den-tal pulp express CD34 antigen and they are MSC.

The SC isolation technique used in most laboratories is based on the use of fl ow-cytometers with cell sorter termed FACS ( fl uorescent activated cell sorter) ( 17– 19 ) . This methodology offers many advantages: high purity; better separation of the popu-lations using multiple parameters on the basis of fl uorescent inten-sity; cell death limited; separation also on the basis of intracellular staining (for examples DNA, expression of cytokines, GFP); from 2 to 6 populations sorted simultaneously; large number of cells examined. This technique, however, presents some important limi-tations: (1) the separation can generate suffering stem cells due to electric charge applied to the cell; (2) semisterility of sample; (3) high cost of cytometer maintenance; (4) high cost of reagents, such as fl uorescent antibodies, and fi nally, (5) long time in the preparation protocols and selection of stem cells. Moreover, the sorters are mainly and only used for high end cellular research. Therefore, new methods/protocols should be proposed to select and isolate stem cells without staining by fl uorescent markers or use of magnetic beads. These new procedures should be based on biophysical differences among the different cell populations in order to obtain interesting peculiarities for implementation in bio-medical/clinical laboratories, such as simplicity and short times of preparation and use of samples, complete sterility of stem cells, the potential disposable, low cost and complete maintenance of the vitality, and integrity of the cells with real-time response for subse-quent applications in the biomedical/clinical/surgical fi elds.

The procedures that are mainly used by researchers in order to detect, isolate, proliferate, and differentiate DPSCs include the following steps:

1. Human dental pulp are extracted from teeth of healthy adults.

2.6. Equipments

3. Methods

3.1. DPSCs Isolation

3.1.1. Dental Pulp Extraction and Digestion


2. Prior to extraction, each subject is checked for systemic and oral infection or diseases.

3. Only disease-free subjects are selected. 4. Each subject, which is usually a patient that must undergo a

third molar extraction, follows a pretreatment with profes-sional dental hygiene.

5. Before extraction, the dental crown is covered with 0.3% chlo-rexidin gel for 2 min and then pulp is extracted with a dentinal excavator or a Gracey curette (Fig. 1 ).

6. Once removed, the pulp is immersed in a digestive solution (3 mg/mL type I collagenase plus 4 mg/mL dispase and gen-tamicin) for 1 h at 37°C in agitation.

7. The solution is then fi ltered with 70- μ m Falcon strainers.

1. After fi ltration, cells are immersed in a culture medium usually the Mega Cell supplemented with 10% FBS, 100 μ M 2P-ascorbic acid, 2 mM L -glutamine, 100 U/mL penicillin, 100 μ g/mL streptomycin (Fig. 2 ), and placed in 75-cm 2 fl asks with fi ltered valves.

2. Flasks are incubated at 37°C and 5% CO 2 and the medium changed twice a week. Just before cells become con fl uent, they can be subdivided into new fl asks.

3. The number of passages ranges from 16 to 20.

3.1.2. Cell Culture

Fig. 1. Extraction of dental pulp with a dentinal excavator from a third molar.


4. Stem cells are sorted (see below) only when their number reaches at least 1,000,000 per fl ask. This number is achieved around day 22, when they are still undifferentiated.

5. Differentiated cells are obtained from sorted stem cells cul-tured for at least 30 days in α -MEM or Mega Cell culture medium with 20% FBS; in fact, FBS exerts a differentiation activity favoring osteoblastic differentiation when used in high percentage ( 8– 11 ) (Fig. 3 ).

1. Cells are detached using 0.02% EDTA solution in PBS and col-lected (10 min at 1,000 rpm), washed in 0.1% BSA in PBS, then incubated in a solution of antibody (1 μ g/ μ L).

3.1.3. Fluorescence-Activated Cell Analysis and Sorting

Fig. 2. Cells after 24 h of culture (original magni fi cation ×100) ( a ); cells at con fl uence after 1 week of culture (original magni fi cation ×100) ( b ).

Fig. 3. Formation of bone calci fi cation nodule from sorted stem cells cultured for at least 30 days in α -MEM at 20% FBS.


2. Cells are then washed in the same solution (see above) and are ready for observation.

3. Antibodies used for sorting are the following mouse anti-human antibodies: CD117 FITC (c-kit), CD34 PE, fl k-1, and STRO-1.

4. We sorted using both morphological (high side scatter and low forward scatter) and antigenic criteria ( fi rst with CD117 and CD34 and then, serially for STRO-1 and fl k-1).

5. Only cells that express all these markers should be selected, in order to obtain a homogeneous population.

6. After sorting, with the same procedures, the cells are analyzed with anti-CD45 CY, anti-CD90 FITC, and anti-CD133 PE.

7. After differentiation, cells are examined for the following anti-bodies, other than the previous antibodies for stem cell mark-ers, in order to assess osteogenic differentiation: CD54, CD44, Osteocalcin, and the transcription factor RUNX-2.

8. For RUNX-2 intracellular analysis, Fix&Perm Kit is used, according to guidelines.

9. To asses endothelial differentiation, the following antibodies are used: CD31 (PECAM-1), von-Willebrand (domain 1 and 2); ACE, and fl k-1.

1. To evaluate colony ef fi ciency and proliferation potential of sorted stem cells, single cells obtained by limiting dilutions are plated.

2. Numbers of clones and cells are evaluated. 3. After 3 weeks of culture, cells are stained with 0.1% (v/v) tolu-

idine blue in 1% paraformaldehyde. 4. The number of clones (>50 cells) are counted.

1. Sorted cells are challenged to assess their multipotency. 2. For smooth muscle differentiation, cells are cultured in the

presence of 2% FBS and 10 ng/mL TGF- β for 4–6 days. 3. For neural differentiation, cells are cultured at high densities,

spontaneously forming spherical clumps of cells that were iso-lated with 0.25% trypsin. Free- fl oating neurospheres that are released from the cell culture surface into the culture media were also collected. The spheres of cells are transferred to a Petri dish and cultured in Neurobasal medium supplemented with B27, 20 ng/mL bFGF, and 20 ng/mL EGF for 4–7 days. The culture density of the spheroid bodies is maintained at 10–20 cells/cm 2 to prevent self-aggregation.

3.1.4. Colony Ef fi ciency Assays and Proliferation Potential

3.1.5. Differentiation


4. For adipocyte differentiation, the culture medium must be supplemented with 10% FBS and 1 μ M dexamethasone, 10 μ M recombinant human insulin, 200 μ M indomethacin, and 0.5 mM 3-isobutyl-1-methyl-xantina (IBMX) twice a week for 2 weeks.

5. Oil red-O staining is used to identify lipid-laden fat cells (Fig. 4 ).

1. Cell cycle is analyzed by fl ow cytometry. 2. Cells are harvested in phosphate-buffered saline (PBS) con-

taining 2 mM EDTA, washed once with PBS, and stained with 50 μ g/mL iodide propide in sodium citrate buffer 0.1 plus 1 mg/mL RNAse A for 2 h at room temperature in the dark.

3. Stained nuclei are analyzed with a fl uorescence-activated cell sorter (FACS) ARIAII and the data analyzed using a Mod-Fit cell cycle analysis program (Fig. 5 ).

4. Analysis is performed in fours for each sample and time from day 15 up to the 45th day of culture.

1. For ALP, cells and tissue samples are washed in PBS and fi xed in 4% paraformaldehyde in PBS, with 0.2% Triton X100 for 30 min at 4°C, then washed twice in 0.1% BSA in PBS at room temperature for 10 min each.

2. ALP activity is measured using 100,000 cells/sample, detached with PBS/EDTA 0.02% and centrifuged for 10 min at 140 × g .

3. The pellet is incubated with 1 mL of BMPurple solution for 8 h in the dark.

4. Supernatant is read in a spectrophotometer at 615 nm. 5. As control, c-kit − /STRO-1 − /CD34 − cells were used.

3.1.6. Cell Cycle Analysis

3.1.7. Histochemistry and Immuno fl uorescence Analyses

Fig. 4. Multivacuolar adipocytes with lipid-laden fat (original magni fi cation, ×100) ( a ); Oil red-O staining (original magni fi cation, ×100) ( b ).


6. The values are expressed as the ratio of sample and BMPurple stock solution.

7. BMPurple solvent was used as blank. 8. For calcein staining, a solution of 50 μ M calcein-AM in PBS is

added to culture medium (1/10 v/v). 9. After incubation at 37°C with 5% CO 2 for 30 min, cells are

washed twice with PBS. 10. The observation is performed using a fl uorescence microscope

with 490-nm excitation and 512-nm emission fi lters. 11. Cells and chips of the new-formed woven bone are fi xed in

2.5% glutaraldehyde in a phosphate buffer. 12. Post fi xed in 0.1% OsO 4 in the same buffered solution for 1 h,

then dehydrated and embedded in epoxy resins. 13. Counterstained (uranyl acetate and lead citrate) ultrathin

sections were observed under an electron microscope (Philips 400 S) (Fig. 6 ).

1. The cells (stocks of 250,000 for stem cells and of 500,000 for differentiated cells) are frozen in 10% DMSO in MegaCell at 10% FBS in a cryotubes and stored for up to 2 years.

2. At the end of the freezing period, cells are quickly thawed by the addition of 1 mL of medium at 10% FBS at 37°C and then added to 10 mL of the same medium.

3. Cells are pelleted at 1,500 rpm for 10 min and the supernatant is removed.

3.1.8. Cryopreservation

Fig. 5. Cell cycle analyses by ModFit software. Percentages of cells in the different cycle phases are indicated and calcu-lated by ModFit software.


4. Fresh medium is added to the tube. Cells are then placed in fl asks and cultured at 37°C in a 5% CO 2 atmosphere.

5. They are vital and the amount of apoptotic cells is less than 10%.

1. Cryopreservation of cells and tissue, mainly of the reproduc-tive system, has been signi fi cantly improved recently, but to date prevailingly hematopoietic stem cells have been cryopre-served and then successfully utilized for transplantation. Moreover, to date there are no reports on the ability of either stem cells or already differentiated cells to restart proliferation, differentiation, and new tissue formation for therapeutic use.

2. After long-term cryopreservation (2 years), osteoblasts differ-entiated from SBP-DPSCs are still capable of quickly restarting proliferation and the production of mineralized matrix, in a manner similar to what we have already demonstrated for fresh cells (see refs. ( 8, 9, 17 ) ). The differences in percentages regarding STRO-1 and fl k-1 with respect to the percentages observed for the other stem cell antigens are due to the fact that we performed multiparametric cell sorting using both

3.2. Cryopreservation of DPSCs

Fig. 6. Osteoblast from dental pulp stem cells.


morphological and antigenic criteria, and sorted fi rst for CD117 and CD34 together and then sequentially for STRO-1 and fl k-1 antigens, before and after cryopreservation (see ref. ( 9 ) ). Thus, preendothelial cells, such as pericytes positive for both CD117 and CD34, could have altered the overall percentages. These cells would be responsible for the differen-tiation of endothelium, which occurs in parallel with osteoblast differentiation, as demonstrated in the embryo during the ossi fi cation process. Moreover, both osteoblasts and endothe-liocytes express the VEGF-2 receptor ( fl k-1).

3. Furthermore, after thawing, no apoptotic death was observed, and cells retained their differentiation multipotency, all of which is of interest when assessing the suitability of stem cells for use after cryopreservation. Moreover, osteoblasts produced a large-scale woven bone, which was observed in at least 100 25-cm 2 fl asks. Samples of this bone, when transplanted into immunosuppressed rats, were remodeled into lamellar bone, further demonstrating their vitality.

4. Ultrastructurally, osteoblasts were cuboidal in shape, forming a layer along the border of the extracellular matrix, as observed in vivo during osteogenesis. These differentiated cells con-tained an extremely diffuse RER as well as matrix membrane vesicles, containing crystal-like structures. These ultrastructural observations con fi rmed that cells were unaltered (see ref. ( 9 ) ).

5. Oppositely, no stem cells can be obtained from whole pulp cryopreservation. Several experiments and efforts were carried out in order to overtake this problem, but without positive results. In fact, when the whole pulp is cryopreserved, stem cells are lost in all cases. Therefore, this technique cannot be followed.

The stem population, isolated and cultured in vitro, includes a mosaicism of different cell types with different stages of differentia-tion. Numerous laboratories use the same techniques of detection and isolation of stem cells: tissue surgical removal under sterile con-ditions, digestion in collagenase/dispase, detection and selection by selective markers. For example, stem cells isolated from dental pulp express CD34 antigen and they are MSC ( 8 ) (see Note 7).

Techniques for cell harvest, culture, expansion, and enrich-ment of stem cells are standardized. Many laboratories use the same techniques based on tissue surgical removal under sterile conditions, transport to a laboratory in a physiological solution, digestion in collagenase/dispase, centrifugation and, then, use of selective markers (depending on cells and origin) using fl uorescence-activated cell sorters (FACS) or magnetic beads. Flow cytometry is a technology that simultaneously measures and then analyzes mul-tiple physical characteristics of single cells, which fl ow in a fl uid

3.3. New Projected Procedures for Stem Cells Isolation


stream through a beam of light. The cell characteristic analyzed include cell’s relative size, granularity or internal complexity, and relative fl uorescence intensity. In recent years, the fl ow cytometry has achieved a remarkable expansion, both in laboratories and in clinical research laboratories. The references to “ fl ow cytometry” in the Medline data base were 0 in 1970, 113 in 1980, 2,286 in 1990, and 4,893 in the year 2000. Factors that have contributed signi fi cantly to this development are: the possibility of using dif-ferent laser emissions, which can be used for multiparametric anal-yses; availability of MoAb conjugated with a wide range of fl uorochromes and direct against a wide variety of membrane/intracellular markers; quantifying several parameters for each indi-vidual cell; detection and isolation of cells by speci fi c marker expression due to evolution of fl ow-cytometers equipped with sorting. Up to now, cell sorting is crucial in the isolation of stem cells starting from heterogeneous cell populations. In a sorting cytometer, cells fl ow through the analysis point where they are illuminated and their scatter and fl uorescence signals detected as in a nonsorting instrument. They then continue to fl ow down-stream where, as the stream breaks up into drops, they become enclosed in individual drops. The fl ow operator will have drawn sort regions around cells “of interest” according to their fl ow parameters. In the case of pulp stem cells, speci fi c stem marker is CD34 antigen ( 20 ) . If a cell in the analysis point has been deter-mined to be a cell of interest according to the sort regions, the drop containing that cell will be charged positively or negatively so that it will be de fl ected either to the left or right as it passes the positive and negative de fl ection plates. Modern cytometers have the ability to charge drops in four ways (strongly or weakly posi-tive and strongly or weakly negative), so that four sort regions can be designated and four subpopulations of cells can be isolated from the original population. Once the stem cell has been isolated from the original population by sorting, it is put in culture to test its differentiation potential, clonogenic and proliferative capacity, and self-renewal.

This methodology offers many advantages, but also some important limitations as discussed above. Therefore, it is impor-tant to develop new protocols to identify and isolate stem cells from tissues. An example could be to realize biosensors, using acoustic resonators, such as thickness-shear mode (TSM) resona-tor, as transducer. In particular, the quartz crystal microbalance (QCM) immunosensor is a popular TSM-based transducer among immunosensing methods. Because of its simplicity, convenience, low cost, and real-time response, this method can be important for detection of biomolecules in the fi eld of cell biology, for the detection and direct differentiation of stem cells to speci fi c cell types.


A QCM is a very sensitive mass deposition sensor based on the piezoelectric properties of the quartz crystal. This technique uses the changes in resonance frequency of the crystal to measure the mass on its surface, because the resonance frequency is highly dependent on any changes of the crystal mass.

QCM have been established as a powerful tools for high sensi-tive measurements of mass, both in biological ( 21 ) and nonbio-logical systems, in air and under vacuum. A QCM is capable of measuring mass deposition down to 0.1 ng. Furthermore, advances in QCM technology, today allows to perform measurements directly in liquid media, by putting the analyzing materials in con-tact with one side of the sensor quartz. Sensitive, selective detec-tion of biochemically active compounds can be achieved by employing antigen–antibody, enzyme–substrate, and other recep-tor protein pairs, several of which have been utilized in the devel-opment of piezoelectric immunoassay devices. In fact, several QCM-based immunological and microbiological methods have been demonstrated and applied, including antibody-based immu-noassay by direct or sandwich methods ( 22, 23 ) , detection of nucleic acids ( 22 ) , glucose detection via hexakinase binding ( 23 ) , real-time measurement of cell metabolism and division rate ( 24 ) , immunological detection of microbes ( 25, 26 ) . The receptor pro-tein (e.g., enzyme, antibody) can be immobilized directly on the sensor surface, or it can be suspended in a suitable fi lm or mem-brane. An example of the sensitivity and response range that can be achieved with a QCM biosensor is provided in Fig. 7 . It should be noted that, in many cases, the extent of af fi nity-based reactions increases with time, so that the detection limit for such biosensors will depend on the analysis time utilized.

3.3.1. QCM Working Principle

Fig. 7. Figure showing an example of the sensitivity and response range achieved with a QCM biosensor. The antibody can be immobilized directly on the sensor surface, and its concentration is directly proportional to the frequency measured by QCM biosensor.


In order to address the issue, it is important to de fi ne a suitable biotechnological approach and deal with some very important technical aspects, like the selection of a suited methodology to attach on the gold surface of quartz crystals antibodies (for exam-ple CD34) directed against speci fi c antigens expressed on the sur-face of selected stem cells.

To fabricate a QCM-based immunosensor, one of the most critical procedures is the electrode modi fi cation. Introduction of a sensing antigen of antibody layer permits the sensor surface to capture the target molecules with high selectivity. Various immobi-lization procedures have been widely used to bind antibodies onto the surface of gold electrodes, including: (a) protein A ( 27, 28 ) (b) polymer membrane ( 29 ) ; (c) covalent attachment via avidin-biotin complex ( 30 ) .

Speci fi cally with antibodies, once immobilized to a solid support, they lose a part of their speci fi c binding competence. This is mainly due to their random orientation on the surface. However, orientated immobilization of antibodies (immunoglobulin G) is possible by using Protein A as a facilitator. This is because immunoglobulin G is made up of three fragments, i.e., two Fab units capable of binding the antigen, and another Fc unit. Protein A speci fi cally recognizes and binds this Fc domain, and hence can be used for oriented immobiliza-tion ( 31, 32 ) . Site-directed immobilization of antibodies on a gold carrier has been described for QCM-based immunosensor ( 33 ) . However, in such systems, the Protein A-coated gold carriers did not allow repeated usage, as the Protein A was not tightly bound to the gold surface. A more reliable immobilization method for antibody binding to Protein A-coated gold surface is therefore required.

Recently, interest in the properties and applications of self-assembled monolayers (SAMs) as methods of protein immobiliza-tion on gold surfaces has grown enormously. SAMs used in QCM modi fi cation offer a method of orientated, covalent attachment of antibodies to the gold surface and can provide a reproducible, ultrathin, densely packed monolayer for the late antibody immobi-lization ( 34, 35 ) . In addition, the covalently bound antibody should be unaffected by exposure to high or low pH, often used in regeneration, thus offering ideal reusable systems ( 36 ) .

This feature offers numerous advantages to sensor performance over the conventional methods, e.g., improvement of detection limits and of the reproducibility of the assay, as well as prevention of nonspeci fi c adsorption.

Various techniques have been used to study the kinetics and structures of SAMs ( 37 ) . Thiols and sul fi des are of particular interest mainly because of their spontaneous chemisorption, reg-ular organization, and high thermal, mechanical, and chemical stability ( 38 ) on gold surfaces. Long chain thiols and sul fi des have been shown to be more thermally stable ( 39 ) . The sensor surface can be activated by using a thiol-containing bifunctional linker.

3.3.2. QCM Functionalization


Reproducible results were obtained with dithiobissuccinimidyl-propionate (DSP) ( 40 ) . DSP forms disul fi de bonds to the gold surface and provides N -hydroxysuccinimide (NHS) groups that can react with the free amino groups on the ligand.

Based on the above-mentioned results reported in the litera-ture, it is necessary to modify the gold surface of quartz crystals using the SAM approach to achieve uniform, stable, and sterically accessible antibody coating for QCM.

In particular, in order to

Obtain highly sensitive sensing surfaces ●

To minimize steric restrictions, to allow the ef fi cient binding ●

of the ligand to the Ab To avoid Ab random orientation on the gold surface ●

Several immobilization strategies must be used, including

1. In this strategy, a more reliable immobilization method for antibody binding onto protein A-coated gold surface.

2. In particular, it is necessary to construct a SAM (made up, for example, of dithiobissuccinimide propionate—DSP) able to covalently bind Protein A, thus generating a modi fi ed gold surface where the Ab may interact perpendicularly to the gold surface ( 41 ) .

3. This serves as a functionalized structure for binding biomole-cules by making a barrier to prevent biomolecules from com-ing into contact with the metal surface.

1. To construct a SAM (made up by a longer chain, i.e., 11-mercaptoundecanoic acid, or 16-mercapto-hexadecanoic acid), where the Abs can be immobilize, through their Lys side chains ( 42 ) . This methodology is also conveniently used for protein immobilization, since Lys are often located on the solvent-exposed surface of biomolecules.

1. A further immobilization methodology foresees the direct covalent bond formation between gold quartz crystal surface and a thiolated antibody ( 43 ) . A thiolation reagent can be used as a cleavable cross-linker, which introduces a sulfhydryl group to an antibody. In particular, modi fi cation of the Ab Lys side chains will generate thiolated functional groups on the Ab sur-face. This functional group will be easily attached covalently to the gold surface, thus generating a monolayer coating.

1. The biotinylation of a biomolecule does not affect its biologi-cal activity, then the avidin-biotin system can work as a bridge to anchor a reagent like an antibody, to an avidin-coated surface. Usually, also in this case, the exposed Lys residues of the Ab are biotinylated.

Gold Surface Ab Binding Mediated by a Protein

Direct Ab Binding on SAM-Modi fi ed Gold Surface Through the Ab Lys Side Chains

Ab Derivatization and Immobilization on Gold Surface

Immobilization via the Avidin-Biotin System


The basis for the detection of human stem cells (HSCs) by the QCM immunosensor are HSC-speci fi c antibodies that are anchored on the surface of the quartz crystals. Currently, antibodies used for the detection and isolation of hematopoetic progenitors are usually directed against the membrane protein CD34. This protein is selectively expressed in early lymphohematopoetic stem and pro-genitor cells in bone marrow and peripheral blood as well as on small-vessel endothelial cells and embryonic fi broblasts.

The protein backbone of CD34 has a molecular weight of just 40 kD. However, the Serin and Threonin-rich extracellular domain of CD34 protein is heavily glycosylated so that the protein runs at a apperent weight of about 110 kD on SDS-gels. In addition to its posttranslationally modi fi ed N-terminus, the protein contains a single transmembrane domain and C-terminal cytoplasmic domain that contains consensus sites for phosphorylation by protein kinase C. The function of CD34 is unkown. It was shown to bind L -selectin and is believed to be involved in cell adhesion and lymphocyte homing.

A number of anti-CD34 antibodies have already been described. Their epitopes are grouped in three distinct classes according to their sensitivity towards digestion with O -sialoglycoprotease from Pasteurella hemolytica or neuraminidase. Class 1 epitopes are sen-sitive to neuraminidase and glycoprotease. These epitopes are not recommended for detection and enumeration of CD34 + cells, as they do not detect all glycoforms of CD34, mostly display low avidity and are instable when conjugated to certain fl uorochromes. Class 2 epitopes are sensitive only to treatment with O -sialoglycoprotease, but not with neuraminidase. Antibodies directed against class 2 epitopes are used in commercial kits for CD34 isolation. A prototype of this epitope class is antibody QBEND10 which detects a linear epitope at the far N-terminus of CD34. Class 3 epitopes are sensitive neither to glyoprotease nor to neuraminidase digestion and can therefore also be used for CD34 + cell enumeration.

The challenge of future research will be focused in order to generate antibodies against class 2 or 3 epitopes that ful fi ll the following conditions:

1. The antibodies need to have a high speci fi city, af fi nity, and avidity for CD34 as to allow selective binding of HSCs.

2. The antibodies need to have a high stability, so that they can be ef fi ciently coupled to the gold surface of the QCM.

3. Previous studies have shown that the binding characteristics of anti-CD34 antibody QBEND10 are altered if the antibody is FITC-labeled. It will therefore be important to compare the binding speci fi cities and af fi nities of antibodies bound to the QCM’s surface to those of unbound antibodies.

Therefore, several techniques should be assayed in parallel, to ensure a successful completion of the new most promising approach.

HSC-Speci fi c Antibodies


1. After weighing, Lyophilized Collagenase and Dispase are dis-solved in PBS, fi ltered by 0.22- μ m syringe fi lters, aliquoted in 50-mL Falcon, and stored at −20°C.

2. FBS is thawed at 37°C in bath, decomplemented at 56°C for 30 min, aliquot in 50-mL Falcon and then frozen at −20°C. When we make the culture medium, we thaw the FBS and then add in the medium.

3. L -glutamine and penicillin and streptomycin are thawed at 37°C in bath and aliquoted in 15-mL Falcon and then refrozen at −20°C.

4. Lyophilized EGF and bFGF are dissolved in H 2 Od at 1% BSA, aliquot in eppendorf, and frozen at −20°C.

5. Lyophilized paraformaldehyde is dissolved in H 2 Od on warmed magnetic stirrer. Concentrated NaOH (1 N) can be used to adjust pH to 7.4. Then, paraformaldehyde at 4% is aliquot in 50-mL Falcon and frozen at −20°C.

6. All antibodies for cytometry are stored at 4°C at the dark until exposure date as indicated by manufacturer.

7. Patents: The whole procedure is protected by the International worldwide Patent “Stem cells obtained from pulp of deciduous or permanent teeth and of dental germ, able to produce human bone tissue” PCT/EP2005/0081; WO 2006/010600 .

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