Comment to: Vegetable peptones as a fetal bovine serum substitute in human deciduous tooth pulp stem cell culture

ABSTRACTObjective This study aimed to evaluate the effects of different concentrations of vegetable peptones (pea, wheat, and soy) as substitutes for fetal bovine serum in stem cell cultures derived from human exfoliated deciduous teeth.Methods Stem cell cultures derived from human exfoliated deciduous teeth were cultured with peptones in different concentrations [0.5%, 1%, and 5% (w/v)] and 10% fetal bovine serum (v/v) as control, and their proliferation was evaluated through the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic differentiation was assessed using Alizarin Red to quantify calcium deposition.Results Wheat, soy, and pea concentrations greater than 1% were cytotoxic to stem cell cultures derived from human exfoliated deciduous teeth. In addition, a long-term study showed that pea peptones were cytotoxic. Studies with soy and wheat peptones were continued at concentrations of 0.5% (w/v), and proliferation on day 3 was greater than 50% compared with the control. Wheat peptone presented more mineralized areas than fetal bovine serum. The aminograms of the three peptones showed that the greater efficiency of wheat peptone may be related to its higher proline and glutamic acid proportions.Conclusion We suggest that vegetable peptones at concentrations ≤1%, particularly 1% wheat, can be used as fetal bovine serum substitutes for stem cell cultures derived from human exfoliated deciduous teeth cultivation.

P93 H2S increases pancreatic regeneration from human tooth pulp

Reply to "comment on: Vegetable peptones as a fetal bovine serum substitute in human deciduous tooth pulp stem cell culture"

Animals are widely used in scientific research as highly specific ‘models’ of humans. Primary cell culture is a widely used model to reduce and replace the use of animal models, as per the 3R concept. We investigated cell stimulatory effects of selected botanical preparations on primary human stem cell cultures, in place of animal models. Primary human fibroblast stem cell (hFSC), human mesenchymal stem cell (hMSC), and human haematopoietic stem cell (hHSC) cultures, were established in-house, and characterized by immunophenotyping. Varying concentrations of selected botanical preparations (mature leaf concentrate of Carica papaya Sri Lankan wild type cultivar [MLCC], distillates of Vernoina/ Mallotus [VMD], and Ficus benghalensis [FBD]) were tested ex vivo for cell proliferation stimulation on these cell platforms, using the MTT assay. Compared with untreated controls, significant proliferative effects were demonstrated by MLCC on female (at 0.2% concentration) and male (0.6%) hMSCs, and on male and female hHSCs (0.6-0.2%) (P<0.05). FBD (0.05-1.2%) stimulated significant proliferation of both male hMSCs, and male hHSCs, but inhibited proliferation of female hMSCs (P<0.05), with no significant effects on female hHSCs. Conversely, VMD (0.2-1%) showed significant inhibitory effects on both male and female hMSCs, yet at 0.6% showed significant proliferative effects on female hHSCs (P<0.05). Selected VMD concentration exhibited approximately 1.5-folds higher % cell stimulation than the positive control (p < 0.01). Thus, eff ective cell proliferative concentrations of botanical preparations were identified using primary stem cell lines, aligning with “replacement” as per the 3R concept. Keywords: Human stem cells; botanical preparations; cell proliferation stimulants; 3R concept; Animal models

Event Abstract Back to Event Human stem cell culture and detachment on biomaterials immobilized with thermoresponsive nanobrush Yi Tung Lu1, Yen Ming Chen1*, Hong Ren Lin1 and Akon Higuchi1, 2* 1 National Central University, Chemical and Materials Engineering, Taiwan 2 King Saud University, Botany and Microbiology, Saudi Arabia Thermoresponsive surface prepared using thermoresponsive polymers with low critical solution temperatures (LCSTs) is attractive candidates for stem cell culture because stem cells can be detached from the surface without applying an enzymatic digestion method and, instead, by decreasing the temperature, which enables cell aggregates or cell sheets to be obtained in culture medium. In this study, we designed the thermoresponsive nano-brush surfaces for human stem cell culture (human adipose-derived stem cells [hADSCs] and human pluripotent stem cells [hPSCs]). Using RAFT polymerization, we prepared the coating copolymers having polystyrene and (a) thermoresponsive poly(N-isopropyl acrylamide), PNIPAAm[1], (b) biocompatible polyethylene glycol methacrylate (PEGMA), and (c) polyacrylic acid (PAA) where bioactive oligopeptide (oligo-vitronectin)[2],[3] can be conjugated via carboxylic acid of PAA. hADSCs were cultured on the surface coated with copolymers containing PNIPAAm, PEGMA and PAA conjugated with oligo-vitronectin. The optimal surface composition where hADSCs can attach and detach by decreasing temperature was investigated. Furthermore, human embryonic stem cells (WA09) and human induced pluripotent stem cells were cultured on the surface coated with these copolymers. We have successfully cultured human stem cells on the thermoresponsive nanobrush surface and can reach 80% cell detachment of hADSCs and 50% cell detachment of hESCs. However, if we could not reach 100% detachment of stem cells, we have another option that thermoresponsive nanobrush surfaces can be reused with partial detachment of stem cells, which do not need the process to reseed cells (passage). We also investigated whether hPSCs could maintain their pluripotency on the surface coated with these copolymers to find out long period (>passage ten) and whether hPSCs could be easily detach from the surface by decreasing the temperature. This results indicate that human stem cells which are highly sensitive to enzymatic treatment can be achieved by used copolymer nanobrush surfaces. Furthermore, we are designing to shift to a novel 3D culture system to scale up for clinical application. Akon Higuchi; Yen Ming Chen; Hong Ren Lin; Saradaprasan Muduli; I Chia Peng; Hsing Fen Li

The aim of this study was to test whether lyophilized conditioned media from human dental pulp mesenchymal stem cell cultures promote the healing of critical-size defects created in the calvaria of rats. Prior to the surgical procedure, the medium in which dental pulp stem cells were cultured was frozen and lyophilized. After general anesthesia, an 8 mm diameter bone defect was created in the calvaria of twenty-four rats. The defects were filled with the following materials: xenograft alone (G1) or xenograft associated with lyophilized conditioned medium (G2). After 14 or 42 days, the animals were euthanized, and the specimens processed for histologic and immunohistochemical analysis. Bone formation at the center of the defect was observed only in the G2 at 42 days. At both timepoints, increased staining for VEGF, a marker for angiogenesis, was observed in G2. Consistent with this, at 14 days, G2 also had a higher number of blood vessels detected by immunostaining with an anti-CD34 antibody. In conclusion, conditioned media from human dental pulp mesenchymal stem cell cultures had a positive effect on the regenerative process in rat critical-size bone defects. Both the formation of bone and enhancement of vascularization were stimulated by the conditioned media.

There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

The current approach to treatment at St. Mary's Hospital of dissecting aortic aneurysms uses a period of induced hypotension before aortography and consideration for surgical correction. There are 13 survivors of a series of 24 patients treated in this way.

Multipotent stem cells from apical pulp of human deciduous teeth with immature apex

The purpose of this study was to investigate the bacterial composition of necrotic pulps of human deciduous teeth by sampling the split surfaces of freshly extracted teeth and culturing the bacteria present with good anaerobic isolation techniques. Significantly more bacteria were recovered after the incubation in an anaerobic chamber than after aerobic incubation in air with 30 per cent CO 2 . Of 276 bacterial isolates, 251 (91 per cent) were obligate anaerobes. These findings suggest that the environment of necrotic pulps in human deciduous teeth is anaerobic and thus favours the growth of anaerobes. Among the 251 obligate anaerobes isolated, strains belonging to the genera Peptostreptococcus (25 per cent), Propionibacterium (19 per cent), Eubacterium (17 per cent) and Fusobacterium (13 per cent) were major parts of the bacterial flora of the lesions of human deciduous teeth. Bifidobacterium (2 per cent), Lactobacillus (1 per cent), Actinomyees (1 per cent) and Veillonella (0.7 per cent) were minor parts of the flora. The microflora of necrotic pulps of human deciduous teeth is in some respects similar to that reported for the deep layers of dentinal lesions of adults. Keywords - Children, Deciduous teeth, Eubacterium , Necrotic pulps, Obligate anaerobes.

Translation: Screening for Novel Therapeutics With Disease-Relevant Cell Types Derived from Human Stem Cell Models

Scientists at Duke University Medical Center have demonstrated they can grow human stem cells in the laboratory by blocking an enzyme that naturally triggers stem cells to mature and differentiate into specialized cells. The discovery may enable scientists to rapidly grow stem cells and transplant them into patients with blood disorders, immune defects and select genetic diseases, said the Duke researchers.Stem cells are the most flexible cells in the body, continually dividing into new stem cells or into specialized cells that carry out specific roles in the body. But little is known about how stem cells choose their fate. The Duke team focused on "hematopoietic" or blood stem cells.In their study, the investigators discovered that an enzyme, aldehyde dehydrogenase (ALDH), stimulates hematopoietic stem cells to mature and transform into blood or immune cells, a process called differentiation. They inhibited this enzyme in stem cell cultures and successfully increased the number of stem cells by 3.4 fold. Moreover, they demonstrated the new stem cells were capable of fully rebuilding the blood-forming and immune systems of immune-deficient mice.Results of the study are published on line and will be published in the August 1, 2006, issue of the Proceedings of the National Academy of Sciences."Our ability to treat human diseases is limited by our knowledge of how human stem cells determine their fate -- that is, whether they maintain their ability to self-renew or whether they go on to become specialized cells," said John Chute, M.D., associate professor of medicine in the Duke Adult Bone Marrow and Stem Cell Transplant Program. "Unraveling the pathways that regulate self-renewal or differentiation in human stem cells can facilitate our ability to expand the growth of human stem cells for therapeutic uses."Currently, patients who require stem cell transplants are given either bone marrow from adult donors, umbilical cord blood derived from newborn babies, or stem cells from blood. But stem cells are scarce, representing less than 0.01 percent of the bone marrow cell population. Likewise, cord blood units frequently lack sufficient numbers of stem cells to rebuild a patient's decimated immune system.Efforts to grow human hematopoietic stem cells in the laboratory have proven extraordinarily difficult, Chute said, because growth factors in culture make stem cells rapidly differentiate. The scientists searched for ways to block a stem cell's natural propensity to differentiate without promoting uncontrolled growth. The researchers focused on the ALDH enzyme because it is a telltale "marker" that distinguishes stem cells from other blood and immune cells. Moreover, it is known to play an essential role in the body's production of retinoic acids, which regulate cell differentiation in a variety of tissues. Yet how ALDH functions in stem cells remained unknown, Chute said.The scientists began by analyzing how stem cells behave under normal circumstances when grown in culture. They mixed together purified human stem cells with growth factors that induce stem cells to mature and differentiate. As expected, the stem cells showed a marked decline in number as they differentiated into other types of specialized cells. By day seven, all stem cells had disappeared from culture.The scientists then added an inhibitor of ALDH to the stem cell cultures, and they found that half of the stem cells maintained their immature and undifferentiated status. Moreover, adding the inhibitor caused a 3.4-fold increase in stem cell numbers within seven days.Next, the scientists transplanted the cultured stem cells into immune-deficient mice to determine how the stem cells would behave. The new population of stem cells migrated to the bone marrow as expected and successfully "engrafted," or took hold in the bone marrow, where they began to produce new blood and immune cells. "ALDH appears to play a fundamental role in the differentiation program of human hematopoietic stem cells," Chute said. "Inhibition of this enzyme facilitates the expansion of human hematopoietic stem cells in culture." Chute said their results reveal a unique role for both ALDH and the process of retinoic acid signaling in human stem cells. Chute and colleague DonaldMcDonnell, Ph.D., professor of pharmacology and cancer biology, are currently testing whether they can directly block the retinoic acid receptors in stem cells and produce a comparable expansion of human stem cells.The investigators plan to develop a clinical trial to test their approach to expand human stem cells for therapeutic purposes.For more information contact: Becky Levine; Tel.: 919.660.1308 or 919.684.4148; Email: levin005@mc.duke.edu

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic ProgenitorFunction and Overcomes Early InVivo Transplantation Failure.

The purpose of this study was to investigate the bacterial composition of necrotic pulps of human deciduous teeth by sampling the split surfaces of freshly extracted teeth and culturing the bacteria present with good anaerobic isolation techniques. Significantly more bacteria were recovered after the incubation in an anaerobic chamber than after aerobic incubation in air with 30 per cent CO2. Of 276 bacterial isolates, 251 (91 per cent) were obligate anaerobes. These findings suggest that the environment of necrotic pulps in human deciduous teeth is anaerobic and thus favours the growth of anaerobes. Among the 251 obligate anaerobes isolated, strains belonging to the genera Peptostreptococcus (25 per cent), Propionibacterium (19 per cent), Eubacterium (17 per cent) and Fusobacterium (13 per cent) were major parts of the bacterial flora of the lesions of human deciduous teeth. Bifidobacterium (2 per cent), Lactobacillus (1 per cent), Actinomyees (1 per cent) and Veillonella (0.7 per cent) were minor parts of the flora. The microflora of necrotic pulps of human deciduous teeth is in some respects similar to that reported for the deep layers of dentinal lesions of adults.