Sabri Musa

Inherent Differential Propensity of Dental Pulp Stem Cells Derived from Human Deciduous and Permanent Teeth

INTRODUCTION Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineage-specific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. METHODS We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. RESULTS The proliferation rate of SCD was higher (cell number, 25 x 10(6) cells/mL; percent colony-forming units [CFUs], 151.67 +/- 10.5; percent cells in S/G2 phase, 12.4 +/- 1.48) than that of DPSCs (cell number, 21 x 10(6) cells/mL; percent CFUs, 133 +/- 17.62; percent cells in S/G2 phase, 10.4 +/- 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (>2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression >100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. CONCLUSIONS This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.

Differentiation of Dental Pulp Stem Cells into Islet-like Aggregates

The post-natal dental pulp tissue contains a population of multipotent mesenchymal progenitor cells known as dental pulp stromal/stem cells (DPSCs), with high proliferative potential for self-renewal. In this investigation, we explored the potential of DPSCs to differentiate into pancreatic cell lineage resembling islet-like cell aggregates (ICAs). We isolated, propagated, and characterized DPSCs and demonstrated that these could be differentiated into adipogenic, chondrogenic, and osteogenic lineage upon exposure to an appropriate cocktail of differentiating agents. Using a three-step protocol reported previously by our group, we succeeded in obtaining ICAs from DPSCs. The identity of ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. There were several-fold up-regulations of these transcription factors proportional to days of differentiation as compared with undifferentiated DPSCs. Day 10 ICAs released insulin and C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Our results demonstrated for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an unconventional and non-controversial source of human tissue that could be used for autologous stem cell therapy in diabetes.

Assessment of Patients' Level of Satisfaction With Cleft Treatment Using the Cleft Evaluation Profile

Objectives: Determination of the psychosocial status and assessment of the level of satisfaction in Malaysian cleft palate patients and their parents. Design: Cross-sectional study. Participants and Methods: Sixty cleft lip and palate patients (12 to 17 years of age) from Hospital Universiti Sains Malaysia and their parents were selected. The questionnaires used were the Child Interview Schedule, the Parents Interview Schedule, and the Cleft Evaluation Profile (CEP), administered via individual interviews. Results: Patients were teased because of their clefts and felt their self-confidence was affected by the cleft condition. They were frequently teased about cleft-related features such as speech, teeth, and lip appearance. Parents also reported that their children were being teased because of their clefts and that their childrens self-confidence was affected by the clefts. Both showed a significant level of satisfaction with the treatment provided by the cleft team. There was no significant difference between the responses of the patients and their parents. The features that were found to be most important for the patients and their parents, in decreasing order of priority, were teeth, nose, lips, and speech. Conclusions: Cleft lip and/or palate patients were teased because of their clefts, and it affected their self-confidence. The Cleft Evaluation Profile is a reliable and useful tool to assess patients’ level of satisfaction with treatment received for cleft lip and/or palate and can identify the types of cleft-related features that are most important for the patients.

Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications

BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbeccos modified Eagles medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth

ObjectivesThe aim of this study was to investigate the immunodulatory properties of dental pulp stem cells derived from healthy (SCD) and inflamed pulp deciduous (SCDIP) tissues. The overall hypothesis is that SCDIP possess equal immune properties with SCD and could be used as an alternative tissue source in regenerative medicine.Materials and methodsAn intra-oral examination was carried out to assess the status of the pulp tissues and group them according to healthy or inflamed. Primary cells were established from these groups, and basic mesenchymal stem cells (MSC) characterizations were conducted. The expression of human leukocyte antigen (HLA), namely HLA-G, HLA-DR, and HLA-ABC were examined in both cell lines using flow cytometry. We further compared the immunosuppressive effects of SCD and SCDIP on phytohemagglutinin-induced T cell proliferation. Supernatants were tested for cytokine profiling using multiplex array.ResultsWhile SCD exhibited typical MSC characteristics, SCDIP on the other hand, did not. Compared with SCDIP, SCD effectively suppresses mitogen-induced T cells proliferation in a dose-dependent manner, as well as express a higher percentage of HLA-ABC and HLA-G. In addition, levels of several cytokines, such as TNF-α, TNF-β, and IL-2, were drastically suppressed in SCD than SCDIP. Furthermore, a high level of IL-10, an important anti-inflammatory cytokine, was present in SCD compared with SCDIP.ConclusionsThese findings suggest that SCDIP is highly dysfunctional in terms of their stemness and immunomodulatory properties.Clinical relevanceSCDIP is not a viable therapeutic cell source especially when used in graft versus host disease (GvHD) and organ rejection.

Unique molecular signatures influencing the biological function and fate of post-natal stem cells isolated from different sources

The discovery of mesenchymal stem cells (MSCs) from a myriad of tissues has triggered the initiative of establishing tailor‐made stem cells for disease‐specific therapy. Nevertheless, lack of understanding on the inherent differential propensities of these cells may restrict their clinical outcome. Therefore, a comprehensive study was done to compare the proliferation, differentiation, expression of cell surface markers and gene profiling of stem cells isolated from different sources, viz. bone marrow, Whartons jelly, adipose tissue and dental pulp. We found that although all MSCs were phenotypically similar to each other, Whartons jelly (WJ) MSCs and dental pulp stem cells (DPSCs) were highly proliferative as compared to bone marrow (BM) MSCs and adipose tissue (AD) MSCs. Moreover, indistinguishable cell surface characteristics and differentiation capacity were confirmed to be similar among all cell types. Based on gene expression profiling, we postulate that BM‐MSCs constitutively expressed genes related to inflammation and immunodulation, whereas genes implicated in tissue development were highly expressed in AD‐MSCs. Furthermore, the transcriptome profiling of WJ‐MSCs and DPSCs revealed an inherent bias towards the neuro‐ectoderm lineage. Based on our findings, we believe that there is no unique master mesenchymal stem cell that is appropriate to treat all target diseases. More precisely, MSCs from different sources exhibit distinct and unique gene expression signatures that make them competent to give rise to specific lineages rather than others. Therefore, stem cells should be subjected to rigorous characterization and utmost vigilance needs to be adopted in order to choose the best cellular source for a particular disease. Copyright

Differential expression of basal microRNAs' patterns in human dental pulp stem cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real‐time PCR. Notably, we observed 19 up‐regulated miRNAs and 29 significantly down‐regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM‐MSCs). The 19 up‐regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa‐miR‐516a‐3p, hsa‐miR‐125b‐1‐3p, hsa‐miR‐221‐5p, hsa‐miR‐7, hsa‐miR‐584‐5p, hsa‐miR‐190a, hsa‐miR‐106a‐5p, hsa‐mir‐376a‐5p, hsa‐mir‐377‐5p and hsa‐let‐7f‐2‐3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa‐miR‐516a‐3p and hsa‐miR‐7‐5p as these miRNAs were highly expressed upon validation with qRT‐PCR analysis. We further proceeded with loss‐of‐function analysis with these miRNAs and we observed that hsa‐miR‐516a‐3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa‐miR‐7‐5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa‐miR‐516a‐3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.

Stem cells conditioned medium: a new approach to skin wound healing management

Stem cell biology has gained remarkable interest in recent years, driven by the hope of finding cures for numerous diseases including skin wound healing through transplantation medicine. Initially upon transplantation, these cells home to and differentiate within the injured tissue into specialised cells. Contrariwise, it now appears that only a small percentage of transplanted cells integrate and survive in host tissues. Thus, the foremost mechanism by which stem cells participate in tissue repair seems to be related to their trophic factors. Indeed, stem cells provide the microenvironment with a wide range of growth factors, cytokines and chemokines, which can broadly defined as the stem cells secretome. In in vitro condition, these molecules can be traced from the conditioned medium or spent media harvested from cultured cells. Conditioned medium now serves as a new treatment modality in regenerative medicine and has shown a successful outcome in some diseases. With the emergence of this approach, we described the possibility of using stem cells conditioned medium as a novel and promising alternative to skin wound healing treatment. Numerous pre‐clinical data have shown the possibility and efficacy of this treatment. Despite this, significant challenges need to be addressed before translating this technology to the bedside.

The Promise of Human Induced Pluripotent Stem Cells in Dental Research

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC lines in vitro from patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

Different isolation methods alter the gene expression profiling of adipose derived stem cells.

Human adipose stem cells (ASCs) has been in the limelight since its discovery as a suitable source of mesenchymal stem cells (MSCs) in regenerative medicine. Currently, two major techniques are used to isolate ASCs, namely liposuction and tissue biopsy. These two methods are relatively risk-free but the question as to which method could give a more efficient output remains unclear. Thus, this study was carried out to compare and contrast the output generated in regards to growth kinetics, differentiation capabilities in vitro, and gene expression profiling. It was found that ASCs from both isolation methods were comparable in terms of growth kinetics and tri-lineage differentiation. Furthermore, ASCs from both populations were reported as CD44+, CD73+, CD90+, CD166+, CD34-, CD45- and HLA-DR-. However, in regards to gene expression, a group of overlapping genes as well as distinct genes were observed. Distinct gene expressions indicated that ASCs (liposuction) has endoderm lineage propensity whereas ASCs (biopsy) has a tendency towards mesoderm/ectoderm lineage. This information suggests involvement in different functional activity in accordance to isolation method. In conclusion, future studies to better understand these gene functions should be carried out in order to contribute in the applicability of each respective cells in regenerative therapy.