Stem cells reside in a quiescent state within all organs of organism in their special niche and they start to proliferate and to migrate when their niche experiences changes 
. Thus, culture of adult SC niche may provide harvesting of SCs at high scale. We developed a method of long-term DP culture, which allowed harvesting of large quantities of IDPSCs (). Mechanical transfer, before and after DP cryopreservation, can also be performed as long as it is necessary and in our experience, it may stop due to, e.g. occasional DP explant contamination. IDPSCs are uniform in respect of morphology (light microscopy and TEM analyzes) () and karyotype of cells remained unchanged (). These cells express high percentage of SC markers such as SH2/CD105, SH3/CD73, nestin, vimentin, fibronectin and low percentage of Oct3/4 (1–F3) without losing their original properties 
To date, standardized protocol of SCs culture from DP is not available. Therefore, we directed our study to optimization culture medium conditions for scaling-up of IDPSCs. DMEM-LG, MEM-alpha and DMEM/F12 are culture media which are commonly used for the isolation and expansion of MSCs 
. In the present work, we verified the effect of DMEM-LG, MEM-alpha and DMEM/F12 on proliferation rate and gene expression pattern of IDPSCs (). These analyses indicated that MEM-alpha and DMEM/F12 were the most appropriate media for the isolation and long-term expansion of IDPSCs (, ); distinct gene expression patterns were observed between these media (). DMEM-LG was not efficient for the isolation, but it was able to support long-term expansion of these cells after their cryopreservation (, ). It seems that our present data (, ) are in contrast with previous observations, which showed IDPSCs exponential growth following multiple passages 
. However, in the present study, cells were counted daily, while in previous study, passages were performed every 3–4 days. Therefore, enzymatic treatment used daily seems to have hampered the IDPSCs.
Analysis of differentiation potential toward chondrogenic and myogenic lineages evidenced high differentiation potential of LP and EP of IDPSCs () comparable to those described previously 
. IDPSCs showed similar chondrogenic and myogenic differentiation before and after cryopreservation (data not shown) as was reported previously for other SCs from DP 
. Cryopreservation process preserves the proliferative and differentiation capacity of IDPSCs and, thus, allows the opportunity to bank these valuable DTSCs 
Recently, new populations of DTSCs were isolated and were shown to be distinct from DPSC (D
ells from permanent teeth)/SHED (S
tem Cells from H
eciduous teeth) 
. As reported in several original publications, DPSC/SHED are supposed to be pericytes, which are isolated from perivascular niche 
. To delineate the anatomic localization of IDPSCs inside the pulp, we performed in situ
analysis using markers of MSCs and ES cells. Our study suggested that DP has multiple SC niches, which are localized in capillaries and nerve networks in cell free zone (); in innermost pulp layer in cell rich zone () and in outermost layer, which contains odontoblasts (). All these niches are rich of nestin positive cells 
, which can present fibroblast-, epithelial- and odontoblast-like morphologies (). In accordance with our finding, recently, rare quiescent multipotent nestin positive MSCs were found in bone marrow in association with hematopoietic SCs and adrenergic nerve fibers. However, bone marrow derived colonies of MSCs cultured in vitro
exhibit a low percentage (~4%) of such nestin positive cells 
. Meanwhile, STRO-1 positive cells localization in DP, as expected, is restricted to endothelium (), albeit very weak positive immunostaining was observed in nervous plexus (). Vimentin showed distribution within DP similar to nestin, however, a smaller amount of cells positive for this markers can be observed (). Overall, these data suggest that IDPSCs constitute a mixed population of both MSCs and epithelial SCs, among which the pericytes (SHED) are also present. Surprisingly, multiple Oct3/4 positive cells were found in DP (), showing the appropriate nuclear localization (). After isolation, IDPSCs did not show as high as expected percentage of Oct3/4 positive cells. Previously, we succeeded to isolate IDPSCs population, which contained ~20% of these cells. We supposed that in the pulp, Oct3/4 positive cells are highly pluripotent and of epithelial type. When these cells start to migrate, they undergo epithelial-mesenchymal transition, which leads to a decrease or lost Oct3/4 expression. Thus, another method can be developed in order to isolate naïve Oct3/4 positive cells from their niche in DP.
DP is capable to produce long-term culture of SCs; however the process of such ability is unknown. BrdU incorporation into DP demonstrates that, as expected, only very rare BrdU positive cells were observed just after DP extraction and plating. Following further cultivation, such cells increased in number and are located in the periphery of DP. It is plausible to suggest, that the isolation of DP stimulates the mechanisms leading to SC proliferation and migration (). In contrast to previous original report 
, which used the method of DP enzymatic dissociation, we choose DP explant as a main method of SCs isolation 
. Comparative morphological analysis demonstrate that prior SCs isolation enzymatic treatment is not recommended for DP which may destroy future “niche” of SCs ().
In conclusion, our method provides the isolation of a high purity SC population in substantial quantities. It is based on natural (intrinsic) mechanisms of SCs activation similar which occur during tissue trauma or injury, when in response to their damage, quiescent SCs are activated. Our method can be applied to isolate SCs from single and multiple niches from any type of adult tissues, such as bone marrow, adipose tissue, umbilical cord, muscles, skin and others. This protocol diminishes a probability of occurrence of spontaneous genomic mutations and eventual karyotype abnormalities, which may arise during multiple passages in SCs. This method is simple, does not requires long time DP preparation, guarantees sterility (DP can be transferred using sterile instruments or even pipette) and avoids any type of SCs selection, which is undesirable for future clinical applications.