Mohammad Mahboob Kanafi

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice.

BACKGROUND AIMS The success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol. METHODS We compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets. RESULTS SHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3-4 weeks, which persisted for >60 days. CONCLUSIONS Our results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.

Midbrain Cues Dictate Differentiation of Human Dental Pulp Stem Cells Towards Functional Dopaminergic Neurons

Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell‐based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell‐types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell‐type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III, and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron‐specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired‐like homeodomain transcription factor 3 (Pitx3) as revealed by real‐time RT‐PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic‐neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1, and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca2+ influx in the presence of KCl, unlike control DPSCs. ATP‐stimulated Ca2+ influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrate for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell‐type. J. Cell. Physiol. 229: 1369–1377, 2014.

Dental pulp stem cells immobilized in alginate microspheres for applications in bone tissue engineering

AIM To immobilize dental pulp stem cells (DPSC) in alginate microspheres and to determine cell viability, proliferation, stem cell characteristics and osteogenic potential of the immobilized DPSCs. METHODOLOGY Human DPSCs isolated from the dental pulp were immobilized in 1% w/v alginate microspheres. Viability and proliferation of immobilized DPSCs were determined by trypan blue and MTT assay, respectively. Stem cell characteristics of DPSCs post immobilization were verified by labelling the cells with CD73 and CD90. Osteogenic potential of immobilized DPSCs was assessed by the presence of osteocalcin. Alizarin red staining and O-cresolphthalein complexone method confirmed and quantified calcium deposition. A final reverse transcriptase PCR evaluated the expression of osteogenic markers - ALP, Runx-2 and OCN. RESULTS More than 80% of immobilized DPSCs were viable throughout the 3-week study. Proliferation appeared controlled and consistent unlike DPSCs in the control group. Presence of CD73 and CD90 markers confirmed the stem cell nature of immobilized DPSCs. The presence of osteocalcin, an osteoblastic marker, was confirmed in the microspheres on day 21. Mineralization assays showed high calcium deposition indicating elevated osteogenic potential of immobilized DPSCs. Osteogenic genes- ALP, Runx-2 and OCN were also upregulated in immobilized DPSCs. Surprisingly, immobilized DPSCs in the control group cultured in conventional stem cell media showed upregulation of osteogenic genes and expressed osteocalcin. CONCLUSION Dental pulp stem cells immobilized in alginate hydrogels exhibit enhanced osteogenic potential while maintaining high cell viability both of which are fundamental for bone tissue regeneration.

Differential Neuronal Plasticity of Dental Pulp Stem Cells from Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons

Based on early occurrence in chronological age, stem‐cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation‐potential toward certain cell‐lineage in comparison to stem‐cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic‐neurons and further, if the difference is reflected in a differential expression of sonic‐hedgehog (SHH)‐receptors and basal‐expressions of tyrosine‐hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain‐cues [SHH, fibroblast growth‐factor8, and basic fibroblast growth‐factor], and their molecular, immunophenotypical, and functional characterization was performed at different time‐points of induction. Though SHED and DPSCs spontaneously expressed early‐neuronal and neural‐crest marker in their naïve state, only SHED expressed a high basal‐expression of TH. The upregulation of dopaminergic transcription‐factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH‐expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular‐Ca2+ influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower‐expression of SHH‐receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down‐regulation of cyclic adenosine‐monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation‐potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic‐neurons than SHED; influenced by higher SHH‐receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048–2063, 2016.

Influence of Hypoxia, High Glucose, and Low Serum on the Growth Kinetics of Mesenchymal Stem Cells from Deciduous and Permanent Teeth

The therapeutic potential of mesenchymal stromal cells depends on their ability to survive and proliferate under adverse in vivo scenarios in a particular disease. In most of the sites of injury, especially in diabetic wounds, there can be hypoxia, hyperglycemia, and ischemia, leading to a lack of nutrients. Hence, the aim of our present study was to investigate the influence of hypoxia, high glucose, and low serum concentrations on the growth kinetics and proliferative potential of human dental pulp stem cells from exfoliated deciduous teeth (SHED) and permanent teeth (DPSC). In this study we isolated two types of specialized stem cells from human dental pulp tissues, which were supposedly of neural crest origin, and cultured them in KO-DMEM medium supplemented with 10% fetal bovine serum (FBS). Both SHED and DPSC were characterized for standard CD surface markers, and their ability to differentiate into adipogenic and osteogenic lineages was tested. SHED and DPSC were exposed to either hypoxia or high glucose or low serum conditions, and their growth kinetics and differentiation potentials were compared with those of normal culture conditions. We found that SHED retained their phenotypic expression and differentiation potential under hypoxia, high-glucose, and low-serum conditions and exhibited a higher proliferation in terms of cell yield and a reduced doubling time compared to DPSC. Our findings clearly demonstrate for the first time that SHED are superior to DPSC as evidenced by their enhanced proliferation under adverse culture conditions.

Phenotypic and functional comparison of optimum culture conditions for upscaling of dental pulp stem cells

Advances in dental pulp stem cell (DPSC) biology and behaviour have promised much in the field of regenerative medicine. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients; however they display diverse characteristics under different culture conditions. Since the success of any stem cell culture is regulated by its own micro‐environment, it is imperative to optimise the growth conditions and establish a generic protocol for maintenance and scale‐up. This study focused on optimisation of long‐term culture conditions of human exfoliated deciduous teeth (SHED) in comparison with DPSCs, employing three commonly used basal media – knockout Dulbeccos modified Eagles medium (KO‐DMEM), α‐MEM and DMEM/F12. Based on their characterisation with respect to morphology, growth kinetics, cell surface marker expression, differentiation capacity and plating density, our findings suggest that cells can be expanded with the highest efficiency in KO‐DMEM medium supplemented with 10% FBS. Additionally, under our standardised xeno‐free (10% human plasma) growth conditions, DPSCs displayed and retained their multipotent attributes until late passages. The differences in the growth and differentiation characteristics between SHED and DPSCs are shown, and certify SHED can be a key element in tissue engineering.

Isolation and Characterization of Human Dental Pulp Stem Cells from Cryopreserved Pulp Tissues Obtained from Teeth with Irreversible Pulpitis.

INTRODUCTION Human dental pulp stem cells (DPSCs) are becoming an attractive target for therapeutic purposes because of their neural crest origin and propensity. Although DPSCs can be successfully cryopreserved, there are hardly any reports on cryopreservation of dental pulp tissues obtained from teeth diagnosed with symptomatic irreversible pulpitis during endodontic treatment and isolation and characterization of DPSCs from such cryopreserved pulp. The aim of this study was to cryopreserve the said pulp tissues to propagate and characterize isolated DPSCs. METHODS A medium consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide was used for cryopreservation of pulp tissues. DPSCs were isolated from fresh and cryopreserved pulp tissues using an enzymatic method. Cell viability and proliferation were determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. DPSC migration and interaction were analyzed with the wound healing assay. Mesenchymal characteristics of DPSCs were verified by flow cytometric analysis of cell surface CD markers. The osteogenic and adipogenic potential of DPSCs was shown by von Kossa and oil red O staining methods, respectively, and the polymerase chain reaction method. RESULT We found no significant difference in CD marker expression and osteogenic and adipogenic differentiation potential of DPSCs obtained from fresh and cryopreserved dental pulp tissue. CONCLUSIONS Our study shows that dental pulp can be successfully cryopreserved without losing normal characteristics and differentiation potential of their DPSCs, thus making them suitable for dental banking and future therapeutic purposes.

Generation of islet-like cell aggregates from human non-pancreatic cancer cell lines

To explore a novel source for the derivation of islets, we examined the differentiation potential of human non-pancreatic cancer cell lines, HeLa (cervical carcinoma cell line) and MCF-7 (breast cancer cell line). These cells were subjected to a serum-free, three-step sequential differentiation protocol which gave two distinct cell populations: single cells and cellular aggregates. Subsequent analysis confirmed their identity as pancreatic acinar cells and islet-like cell aggregates (ICAs), as evidenced by amylase secretion and diphenylthiocarbazone staining respectively. Reverse transcriptase-PCR and immunocytochemistry assessment of the ICAs revealed the expression of pancreatic specific markers Ngn-3, Glut-2, Pax-6 and Isl-1. These ICAs secreted insulin in response to glucose challenge, confirming their functionality. We propose that ICAs generated from HeLa and MCF-7 cell lines could form a promising in vitro platform of human islet equivalents (hIEQs) for diabetes research.

Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.

Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT–PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin–EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing.

Modulus-dependent characteristics of Wharton’s jelly mesenchymal stem cells (WJMSC) encapsulated in hydrogel microspheres

Recent studies revealing stem cell behavior dependence on mechanical properties of a substrate has initiated the need to probe matrix mechanics and its influence on stem cell fate in a physiologically relevant three-dimensional (3D) microenvironment. We investigated the proliferative and osteogenic potentials of Wharton’s jelly mesenchymal stem cells (WJMSCs) immobilized in alginate microspheres with respect to the mechanical properties of alginate hydrogels (1, 1.5 and 2% (w/v)) post incubation in a simulated in vivo environment. Compressive moduli, degradation profile, and swelling kinetics of the hydrogels varied proportionally with alginate concentration and with exposure to simulated conditions. Degradation profile and morphological analysis showed that hydrogels exhibiting high modulus (2% w/v) remained the most intact at the end of day 21. High cell viability in all conditions was observed throughout the culture period. Low-modulus hydrogels (1% w/v) facilitated proliferation of WJMSCs whereas high-modulus hydrogels demonstrated better osteogenic differentiation inferred by an up regulation of osteo-specific genes, expressions of osteocalcin, and quantification of calcium deposition. These findings present a step forward in the development of application-specific hydrogel matrices for stem cell-based tissue engineering.