Transgene-free induced pluripotent dental stem cells for neurogenic differentiation

A stem-cell-based therapy could be the ultimate strategy for the regeneration of degenerated nervous tissues. While neural progenitor cells are limited, the generation of functional nervous tissue cells from non-neural somatic cells (for example, dental stem cells) is highly desired. The recent publication in Stem Cell Research and Therapy by Huang and colleagues is an interesting contribution to this topic. The present commentary puts this paper in context with contemporary reports about (transgene-free) induced pluripotent stem cells and neurogenic differentiation.

Th e generation of neural tissue cells is the ultimate goal for stem-cell-based therapies of currently untreatable neurodegenerative diseases such as Parkinson's disease or multiple sclerosis. To date the production of suffi cient numbers of functional neurons or glial cells from nonneural stem/progenitor cells is diffi cult to achieve. A recent study by George Huang and colleagues published in Stem Cell Research and Th erapy used stem cells from the dental apical papilla (SCAP) [1]. Th ese neural-crestderived dental cells express typical neural cell markers such as β-III-tubulin and are closely related to the neuralcrest-derived cells of the peripheral nervous system. SCAP, like other types of dental stem cells, are therefore a favorable cell source for therapies of degenerated nervous tissues. Although neurogenic diff erentiation occurs, protocols for dental stem cells are sophisticated and the neurogenic diff eren tiation is less complete than that of neuroectodermal stem/progenitor cells [2][3][4]. Moreover, the use of SCAP for the regeneration of nervous tissues is also proble matic, because numbers of stem cells are limited in dental apical papillae. Huang and colleagues' new publication has tackled this problem with transgenefree (TF) induced pluripotent stem cells (iPSCs) from SCAP [1].
An appropriate strategy to improve the proliferation and the diff erentiation potential of somatic (stem) cells is the establishment of iPSCs with similar functional and molecular phenotypic characteristics to embryonic stem cells (ESCs) [5]. Th e generation of iPSCs from somatic cells was a decisive step for the direction of stem cell research, and it is no coincidence that the father of iPSCs, Shinya Yamanaka, received the Nobel Prize for this achieve ment [6]. In an earlier study by Huang and colleagues, three types of dental stem cells -SCAP, dental pulp stem cells and stem cells from human exfoliated deciduous teeth -were easily reprogrammed into iPSCs at a higher reprogramming rate than dermal fi broblasts [7]. Although these dental iPSCs had typical characteristics of ESCs, they did also have unfavorable features. For example, most of the frozen-down dental iPSCs did not survive after thawing or the dental iPSCs underwent massive cell death after diff erentiation toward mesenchymal cell lineages with an ESC standard diff erentiation protocol [1,7].
In the present study Huang and coworkers speculated that a permanent integration of viral vectors in iPSCs with a constitutive transgene expression may contribute to the unfavorable features of dental iPSCs [1]. Th ey therefore generated TF iPSCs with a single lentiviral stem cell cassette fl anked by a loxP site (hSTEMCCA-LoxP) vector [1,8]. Two years ago, Sommer and colleagues estab lished this cre-recombinase excisable lentiviral stem cell cassette with an effi ciency to obtain hundreds of iPSCs from a single starting 35-mm plate of human dermal fi broblasts [8]. Th e effi ciency for the generation of TF iPSCs with this method is much higher than that of other protocols; for example, strategies with recombinant proteins [9]. Although a low theoretical risk for insertional mutagenesis remains after cre-recombinase excision, the article by Huang and colleagues showed that the use of the hSTEMCCA-LoxP vector is an appropriate Abstract A stem-cell-based therapy could be the ultimate strategy for the regeneration of degenerated nervous tissues. While neural progenitor cells are limited, the generation of functional nervous tissue cells from non-neural somatic cells (for example, dental stem cells) is highly desired. The recent publication in Stem Cell Research and Therapy by Huang and colleagues is an interesting contribution to this topic. The present commentary puts this paper in context with contemporary reports about (transgene-free) induced pluripotent stem cells and neurogenic diff erentiation.

© 2010 BioMed Central Ltd
Transgene-free induced pluripotent dental stem cells for neurogenic diff erentiation strategy for the reprogramming of dental stem cells [1]. Th e TF SCAP iPSCs, for example, were able to recover better after freezing/thawing, they were able to diff erentiate into mesenchymal cell lineages without cell death and, most importantly, the embryonic bodymediated neurogenic diff erentiation was successful [1].
Surprisingly, TF SCAP iPSCs expressed neural cell markers even without the induction of neurogenic diff erentiation [1]. Th e expression of neural cell markers suggests that TF SCAP iPSCs could have retained residual features of their neural crest cell origin. General variations of iPSCs from diff erent donor cells are known that are probably caused by an incomplete reset of the tissue-specifi c epigenetic memory [10]. Although the actual induction of pluripotency is successful, residual retained imprinting variations of donor cells may also have an impact on the quality of iPSC diff erentiation potentials. For example, keratinocyte-derived iPSCs showed an enhanced keratinocyte potential relative to cord blood-derived iPSCs [11]. Moreover, the neurogenic diff erentiation of iPSCs is variable in comparison with that of ESCs [12]. Th e preferential neurogenic diff er entiation potential of TF SCAP iPSCs has not been established, but the absence of the glial cell marker glial fi brillary acidic protein in both diff erentiated and undiff er entiated TF SCAP iPSCs may favor a diff eren tiation similar to that of ordinary dental stem cells [2,3].
Th e induction of neural cell markers may also consider a relation of TF SCAP iPSCs to the recently established induced neural progenitor cells (iNPCs) [13]. A transient expression of the reprogramming factors, which were also the reprogramming factors for TF SCAP iPSCs, could effi ciently transdiff erentiate fi broblasts into functional iNPCs. Here, fi broblasts were cultivated in two diff erent cell culture media including a specifi c serumfree neural stem cell reprogramming medium [13]. In con trast, in the recent study by Huang and colleagues the reprogramming and the subsequent neural diff erentiation were achieved by the induction of a reprogrammed pluripotent state. TF SCAP iPSCs expressed Oct4, which is one of the most specifi c factors of pluripotency [1]. Th is initial diff eren tiation was followed by diff erentiation into embryonic bodies and later into neural-like cells with a specifi c neurogenic diff erentiation medium for pluripotent stem cells. However, undiff erentiated and neuro genic diff erentiated TF SCAP iPSCs are possibly related to iNPCs. Further elaborating experiments are required to evaluate the nature of TF SCAP iPSCs and their relation to iNPCs.
In conclusion, although the results of this work are promising, we cannot foresee whether a strategy employing iPSCs will be the optimal strategy for cellular therapies with dental stem cells. Dental stem cells (SCAP, dental pulp stem cells or dental follicle cells) are neural crest derived and as such they have a reasonable neurogenic diff erentiation potential [2][3][4]. Nevertheless, to determine the optimal strategy, the neurogenic diff erentiation potential of dental stem cells needs further evaluation. TF SCAP iPSCs from this work should be involved in these new investigations.