Search results for: stem cell

Authors: K. Barbaro, F. Di Egidio, A. Amaddeo, G. Lupoli, S. Eramo, G. Barraco, D. Amaddeo, C. Gallottini

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In this study, we wanted to evaluate the effects of nano-hydroxy apatite (NHA) on mesenchymal stem cells extracted from subcutaneous adipose tissue of the dog. The stem cells were divided into 6 experimental groups at different concentrations of NHA. The comparison was made with a control group of stem cell grown in standard conditions without NHA. After 1 week, the cells were fixed with 10% buffered formalin for 1 hour at room temperature and stained with Giemsa, measured at the inverted optical microscope. The morphological evaluation of the control samples and those treated showed that stem cells adhere to the substrate and proliferate in the presence of nanohydroxy apatite at different concentrations showing no detectable toxic effects.

Keywords: nano-hydroxy apatite, adipose mesenchymal stem cells, dog, morphological evaluation

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Authors: Varsha Salian, Shama Rao, N. Narendra, B. Mohana Kumar

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Evolving evidence proposes the existence of a highly tumorigenic subpopulation of undifferentiated, self-renewing cancer stem cells, responsible for exhibiting resistance to conventional anti-cancer therapy, recurrence, metastasis and heterogeneous tumor formation. Importantly, the mechanisms exploited by cancer stem cells to resist chemotherapy are very less understood. Oral squamous cell carcinoma (OSCC) is one of the most regularly diagnosed cancer types in India and is associated commonly with alcohol and tobacco use. Therefore, the isolation and in vitro characterization of cancer stem-like cells from patients with OSCC is a critical step to advance the understanding of the chemoresistance processes and for designing therapeutic strategies. With this, the present study aimed to establish and characterize cancer stem-like cells in vitro from OSCC. The primary cultures of cancer stem-like cell lines were established from the tissue biopsies of patients with clinical evidence of an ulceroproliferative lesion and histopathological confirmation of OSCC. The viability of cells assessed by trypan blue exclusion assay showed more than 95% at passage 1 (P1), P2 and P3. Replication rate was performed by plating cells in 12-well plate and counting them at various time points of culture. Cells had a more marked proliferative activity and the average doubling time was less than 20 hrs. After being cultured for 10 to 14 days, cancer stem-like cells gradually aggregated and formed sphere-like bodies. More spheroid bodies were observed when cultured in DMEM/F-12 under low serum conditions. Interestingly, cells with higher proliferative activity had a tendency to form more sphere-like bodies. Expression of specific markers, including membrane proteins or cell enzymes, such as CD24, CD29, CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1) is being explored for further characterization of cancer stem-like cells. To summarize the findings, the establishment of OSCC derived cancer stem-like cells may provide scope for better understanding the cause for recurrence and metastasis in oral epithelial malignancies. Particularly, identification and characterization studies on cancer stem-like cells in Indian population seem to be lacking thus provoking the need for such studies in a population where alcohol consumption and tobacco chewing are major risk habits.

Keywords: cancer stem-like cells, characterization, in vitro, oral squamous cell carcinoma

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Authors: Nurhayati Abdullah, Fauziah Sulaiman, Muhamad Azman Miskam, Rahmad Mohd Taib

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Banana pseudo-stem and fruit-bunch-stem are agricultural residues that can be used for conversion to bio-char, bio-oil, and gases by using thermochemical process. The aim of this work is to characterize banana pseudo-stem and banana fruit-bunch-stem through proximate analysis, elemental analysis, chemical analysis, thermo-gravimetric analysis, and heating calorific value. The ash contents of the banana pseudo-stem and banana fruit-bunch-stem are 11.0 mf wt.% and 20.6 mf wt.%; while the carbon content of banana pseudo-stem and fruit-bunch-stem are 37.9 mf wt.% and 35.58 mf wt.% respectively. The molecular formulas for banana stem and banana fruit-bunch-stem are C24H33NO26 and C19H29NO33 respectively. The measured higher heating values of banana pseudo-stem and banana fruit-bunch-stem are 15.5MJ/kg and 12.7 MJ/kg respectively. By chemical analysis, the lignin, cellulose, and hemicellulose contents in the samples will also be presented. The feasibility of the banana wastes to be a feedstock for thermochemical process in comparison with other biomass will be discussed in this paper.

Keywords: banana waste, biomass, renewable energy, thermo-chemical characteristics

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Authors: Armin Khaghani Boroujeni, Leila Dehghani, Parham Talebi Boroujeni, Sahar Rostamian, Ali Asilian

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Background: Hair loss is a common complaint observed in both genders. Androgenetic alopecia is known pattern for hair loss. To assess new regenerative strategies (PRP, A-SC-BT, conditioned media, exosome-based treatments) compared to conventional therapies for hair loss or hair regeneration, an updated review was undertaken. To address this issue, we carried out this systematic review to comprehensively evaluate the efficacy of cell-based therapies on hair loss. Methods: The available online databases, including ISI Web of Science, Scopus, and PubMed, were searched systematically up to February 2022. The quality assessment of included studies was done using the Cochrane Collaboration's tool. Results: As a result, a total of 90 studies involving 2345 participants were included in the present study. The enrolled studies were conducted between 2010 and 2022. The subjects’ mean age ranged from 19 to 55.11 years old. Approaches using platelet rich plasma (PRP) provide a beneficial impact on hair regrowth. However, other cell-based therapies, including stem cell transplant, stem cell-derived conditioned medium, and stem cell-derived exosomes, revealed conflicting evidence. Conclusion: However, cell-based therapies for hair loss are still in their infancy, and more robust clinical studies are needed to better evaluate their mechanisms of action, efficacy, safety, benefits, and limitations. In this review, we provide the resources to the latest clinical studies and a more detailed description of the latest clinical studies concerning cell-based therapies in hair loss.

Keywords: cell-based therapy, exosome, hair restoration, systematic review

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Authors: Halima Albalushi, Mohadese Boroojerdi, Murtadha Alkhabori

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Introduction: Maintenance of stem cell properties during culture necessitates the recreation of the natural cell niche. Studies reported the promising outcome of mesenchymal stem cells (MSC) properties maintenance after using extracellular matrix such as CELLstart™, which is the recommended coating material for stem cells cultured in serum-free and xeno-free conditions. Laminin-521 is known as a crucial adhesion protein, which is found in natural stem cell niche, and plays an important role in facilitating the maintenance of self-renewal, pluripotency, standard morphology, and karyotype of human pluripotent stem cells (PSCs). The aim of this study is to investigate the effects of Laminin-521 on human umbilical cord-derived mesenchymal stem cells (UC-MSC) characteristics as a step toward clinical application. Methods: Human MSC were isolated from the umbilical cord via the explant method. Umbilical cord-derived-MSC were cultured in serum-free and xeno-free conditions in the presence of Laminin-521 for six passages. Cultured cells were evaluated by morphology and expansion index for each passage. Phenotypic characterization of UC-MSCs cultured on Laminin-521 was evaluated by assessment of cell surface markers. Results: Umbilical cord derived-MSCs formed small colonies and expanded as a homogeneous monolayer when cultured on Laminin-521. Umbilical cord derived-MSCs reached confluence after 4 days in culture. No statistically significant difference was detected in all passages when comparing the expansion index of UC-MSCs cultured on LN-521 and CELLstart™. Phenotypic characterization of UC-MSCs cultured on LN-521 using flow cytometry revealed positive expression of CD73, CD90, CD105 and negative expression of CD34, CD45, CD19, CD14 and HLA-DR.Conclusion: Laminin-521 is comparable to CELLstart™ in supporting UC-MSCs expansion and maintaining their characteristics during culture in xeno-free and serum-free culture conditions.

Keywords: mesenchymal stem cells, culture, laminin-521, xeno-free serum-free

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Authors: Brian M. Mehling, Louis Quartararo, Marine Manvelyan, Paul Wang, Dong-Cheng Wu

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Numerous investigations suggest that Mesenchymal Stem Cells (MSCs) in general represent a valuable tool for therapy of symptoms related to chronic inflammatory diseases. Blue Horizon Stem Cell Therapy Program is a leading provider of adult and children’s stem cell therapies. Uniquely we have safely and efficiently treated more than 600 patients with documenting each procedure. The purpose of our study is primarily to monitor the immune response in order to validate the safety of intravenous infusion of human umbilical cord blood derived MSCs (UC-MSCs), and secondly, to evaluate effects on biomarkers associated with chronic inflammation. Nine patients were treated for conditions associated with chronic inflammation and for the purpose of anti-aging. They have been given one intravenous infusion of UC-MSCs. Our study of blood test markers of 9 patients with chronic inflammation before and within three months after MSCs treatment demonstrates that there is no significant changes and MSCs treatment was safe for the patients. Analysis of different indicators of chronic inflammation and aging included in initial, 24-hours, two weeks and three months protocols showed that stem cell treatment was safe for the patients; there were no adverse reactions. Moreover data from follow up protocols demonstrates significant improvement in energy level, hair, nails growth and skin conditions. Intravenously administered UC-MSCs were safe and effective in the improvement of symptoms related to chronic inflammation. Further close monitoring and inclusion of more patients are necessary to fully characterize the advantages of UC-MSCs application in treatment of symptoms related to chronic inflammation.

Keywords: chronic inflammatory diseases, intravenous infusion, stem cell therapy, umbilical cord blood derived mesenchymal stem cells (UC-MSCs)

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Authors: Ainur Sharip, Jose E. Perez, Nouf Alsharif, Aldo I. M. Bandeas, Enzo D. Fabrizio, Timothy Ravasi, Jasmeen S. Merzaban, Jürgen Kosel

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Bone marrow-derived human mesenchymal stem cells (MSCs) are attractive candidates for tissue engineering and regenerative medicine, due to their ability to differentiate into osteoblasts, chondrocytes or adipocytes. Differentiation is influenced by biochemical and biophysical stimuli provided by the microenvironment of the cell. Thus, altering the mechanical characteristics of a cell culture scaffold can directly influence a cell’s microenvironment and lead to stem cell differentiation. Mesenchymal stem cells were cultured on densely packed, vertically aligned magnetic iron nanowires (NWs) and the effect of NWs on the cell cytoskeleton rearrangement and differentiation were studied. An electrochemical deposition method was employed to fabricate NWs into nanoporous alumina templates, followed by a partial release to reveal the NW array. This created a cell growth substrate with free-standing NWs. The Fe NWs possessed a length of 2-3 µm, with each NW having a diameter of 33 nm on average. Mechanical stimuli generated by the physical movement of these iron NWs, in response to a magnetic field, can stimulate osteogenic differentiation. Induction of osteogenesis was estimated using an osteogenic marker, osteopontin, and a reduction of stem cell markers, CD73 and CD105. MSCs were grown on the NWs, and fluorescent microscopy was employed to monitor the expression of markers. A magnetic field with an intensity of 250 mT and a frequency of 0.1 Hz was applied for 12 hours/day over a period of one week and two weeks. The magnetically activated substrate enhanced the osteogenic differentiation of the MSCs compared to the culture conditions without magnetic field. Quantification of the osteopontin signal revealed approximately a seven-fold increase in the expression of this protein after two weeks of culture. Immunostaining staining against CD73 and CD105 revealed the expression of antibodies at the earlier time point (two days) and a considerable reduction after one-week exposure to a magnetic field. Overall, these results demonstrate the application of a magnetic NW substrate in stimulating the osteogenic differentiation of MSCs. This method significantly decreases the time needed to induce osteogenic differentiation compared to commercial biochemical methods, such as osteogenic differentiation kits, that usually require more than two weeks. Contact-free stimulation of MSC differentiation using a magnetic field has potential uses in tissue engineering, regenerative medicine, and bone formation therapies.

Keywords: cell substrate, magnetic nanowire, mesenchymal stem cell, stem cell differentiation

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Authors: Lay Poh Tan, Chor Yong Tay, Haiyang Yu

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Micro-contact printing is a form of soft lithography that uses the relief patterns on a master polydimethylsiloxane (PDMS) stamp to form patterns of self-assembled monolayers (SAMs) of ink on the surface of a substrate through conformal contact technique. Here, we adopt this method to print proteins of different dimensions on our biodegradable polymer substrates. We started off with printing 20-500 μm scale lanes of fibronectin to engineer the shape of bone marrow derived human mesenchymal stem cell (hMSCs). After 8 hours of culture, the hMSCs adopted elongated shapes, and upon analysis of the gene expressions, genes commonly associated with myogenesis (GATA-4, MyoD1, cTnT and β-MHC) and neurogenesis (NeuroD, Nestin, GFAP, and MAP2) were up-regulated but gene expression associated to osteogenesis (ALPL, RUNX2, and SPARC) were either down modulated or remained at the nominal level. This is the first evidence that cellular morphology control via micropatterning could be used to modulate stem cell fate without external biochemical stimuli. We further our studies to modulate the focal adhesion (FA) instead of the macro shape of cells. Micro-contact printed islands of different smaller dimensions were investigated. We successfully regulated the FAs into dense FAs and elongated FAs by micropatterning. Additionally, the combined effects of hard (40.4 kPa), and intermediate (10.6 kPa) PA gel and FAs patterning on hMSCs differentiation were studied. Results showed that FA and matrix compliance plays an important role in hMSCs differentiation, and there is a cross-talk between different physical stimulants and the significance of these stimuli can only be realized if they are combined at the optimum level.

Keywords: micro-contact printing, polymer substrate, cell-material interaction, stem cell differentiation

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Authors: Kirsten Wilson, Patrick M. Brauer, Sandra Babic, Diana Golubeva, Jessica Van Eyk, Tinya Wang, Avanti Karkhanis, Tim A. Le Fevre, Andy I. Kokaji, Allen C. Eaves, Sharon A. Louis, , Nooshin Tabatabaei-Zavareh

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Long-lived plasma cells are rare, non-proliferative B cells generated from antibody-secreting cells (ASCs) following an immune response to protect the host against pathogen re-exposure. Despite their therapeutic potential, the lack of in vitro protocols in the field makes it challenging to use B cells as a cellular therapeutic tool. As a result, there is a need to establish robust and reproducible methods for the generation of B cells. To address this, we have developed a culture system for generating B cells from hematopoietic stem and/or progenitor cells (HSPCs) derived from human umbilical cord blood (CB) or pluripotent stem cells (PSCs). HSPCs isolated from CB were cultured using the StemSpan™ B Cell Generation Kit and produced CD19+ B cells at a frequency of 23.2 ± 1.5% and 59.6 ± 2.3%, with a yield of 91 ± 11 and 196 ± 37 CD19+ cells per input CD34+ cell on culture days 28 and 35, respectively (n = 50 - 59). CD19+IgM+ cells were detected at a frequency of 31.2 ± 2.6% and were produced at a yield of 113 ± 26 cells per input CD34+ cell on culture day 35 (n = 50 - 59). The B cell receptor loci of CB-derived B cells were sequenced to confirm V(D)J gene rearrangement. ELISpot analysis revealed that ASCs were generated at a frequency of 570 ± 57 per 10,000 day 35 cells, with an average IgM+ ASC yield of 16 ± 2 cells per input CD34+ cell (n = 33 - 42). PSC-derived HSPCs were generated using the STEMdiff™ Hematopoietic - EB reagents and differentiated to CD10+CD19+ B cells with a frequency of 4 ± 0.8% after 28 days of culture (n = 37, 1 embryonic and 3 induced pluripotent stem cell lines tested). Subsequent culture of PSC-derived HSPCs increased CD19+ frequency and generated ASCs from 1 - 2 iPSC lines. This method is the first report of a serum- and feeder-free system for the generation of B cells from CB and PSCs, enabling further B lineage-specific research for potential future clinical applications.

Keywords: stem cells, B cells, immunology, hematopoiesis, PSC, differentiation

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Authors: Aditi Mittal, Nishit Gupta, Tina Dadu, Anil Handoo

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Introduction: Hematopoietic stem cell transplantation (HSCT) is a curative treatment done for hematologic malignancies and other clinical conditions. Its main objective is to reconstitute the hematopoietic system of the recipient by administering an infusion of donor hematopoietic stem cells. Transplant engraftment is the first sign of bone marrow recovery. The main objective of this study is to assess immature platelet fraction (IPF) and immature reticulocyte fraction (IRF) as early indicators of post-hematopoietic stem cell transplant engraftment. Methods: Patients of all age groups and both genders undergoing both autologous and allogeneic transplants were included in the study. All the CBC samples were run on Mindray CAL-8000 (BC-6800 plus; Shenzhen, China) analyser and assessed for IPF and IRF. Neutrophil engraftment was defined as the first of three consecutive days with an ANC >0.5 x 109/L and platelet engraftment with a count >20 x 109/L. The cut-off values for IRF were calculated as 13.5% with a CV of 5% and for IPF was 19% with a CV of 12%. Results: The study sample comprised 200 patients, of whom 116 had undergone autologous HSCT, and 84 had undergone allogeneic HSCT. We observed that IRF anticipated the neutrophil recovery by an average of 5 days prior to IPF. Though there was no significant variation in IPF and IRF for the prediction of platelet recovery, IRF was preceded by 1 or 2 days to IPF in 25% of cases. Conclusions: Both IPF and IRF can be used as reliable parameters as predictors for post-transplant engraftment; however, IRF seems to be more reliable than IPF as a simple, inexpensive, and widely available tool for predicting marrow recovery several days before engraftment.

Keywords: transplantation, stem cells, reticulocyte, engraftment

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Authors: Aliya Sekenova, Vyacheslav Ogay

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The derivation of human induced pluripotent stem cells (iPSCs) from somatic cells by direct reprogramming opens wide perspectives in the regenerative medicine. It means the possibility to develop the personal and, consequently, any immunologically compatible cells for applications in cell-based therapy. The purpose of our study was to develop the technology for the production of NK cells from T cell-derived induced pluripotent stem cells (TiPSCs) for subsequent application in adoptive cancer immunotherapy. Methods: In this study iPSCs were derived from peripheral blood T cells using Sendai virus vectors expressing Oct4, Sox2, Klf4 and c-Myc. Pluripotent characteristics of TiPSCs were examined and confirmed with alkaline phosphatase staining, immunocytochemistry and RT-PCR analysis. For NK cell differentiation, embryoid bodies (EB) formed from (TiPSCs) were cultured in xenogeneic serum-free medium containing human serum, IL-3, IL-7, IL-15, SCF, FLT3L without using M210-B4 and AFT-024 stromal feeder cells. After differentiation, NK cells were characterized with immunofluorescence analysis, flow cytometry and cytotoxicity assay. Results: Here, we for the first time demonstrate that TiPSCs can effectively differentiate into functionally active NK cells without M210-B4 and AFT-024 xenogeneic stroma cells. Immunofluorescence and flow cytometry analysis showed that EB-derived cells can differentiate into a homogeneous population of NK cell expressing high levels of CD56, CD45 and CD16 specific markers. Moreover, these cells significantly express killing activation receptors such as NKp44 and NKp46. In the comparative analysis, we observed that NK cells derived using feeder-free culture system have more high killing activity against K-562 tumor cells, than NK cells derived by feeder-dependent method. Thus, we think that our obtained data will be useful for the development of large-scale production of NK cells for translation into cancer immunotherapy.

Keywords: induced pluripotent stem cells, NK cells, T cells, cell diffentiation, feeder cell-free culture system

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Authors: Ahmed Gaber Abdel Raheem, Nashwa Ahmed Mohamed

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Introduction: Sensorineural hearing loss (SNHL) is largely caused by the degeneration of the cochlea. Therapeutic options for SNHL are limited to hearing aids and cochlear implants. The cell transplantation approach to the regeneration of hair cells has gained considerable attention because stem cells are believed to accumulate in the damaged sites and have the potential for the repair of damaged tissues. The aim of the work: was to assess the use of bone marrow transplantation in repair of damaged inner ear hair cells in rats after the damage had been inflicted by Amikacin injection. Material and Methods: Thirty albino rats were used in this study. They were divided into three groups. Each group ten rats. Group I: used as control. Group II: Were given Amikacin- intratympanic injection till complete loss of hearing function. This could be assessed by Distortion product Otoacoustic Emission (DPOAEs) and / or auditory brain stem evoked potential (ABR). GroupIII: were given intra-peritoneal injection of bone marrow stem cell after complete loss of hearing caused by Amikacin. Clinical assessment was done using DPOAEs and / or auditory brain stem evoked potential (ABR), before and after bone marrow injection. Histological assessment of the inner ear was done by light and electron microscope. Also, Detection of stem cells in the inner ear by immunohistochemistry. Results: Histological examination of the specimens showed promising improvement in the structure of cochlea that may be responsible for the improvement of hearing function in rats detected by DPOAEs and / or ABR. Conclusion: Bone marrow stem cells transplantation might be useful for the treatment of SNHL.

Keywords: amikacin, hair cells, sensorineural hearing loss, stem cells

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Authors: Debabrata Auddya, Bradley J. Roth

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The mechanical bidomain model is used to describe a colony of cells growing on a substrate. Analytical expressions are derived for the intracellular and extracellular displacements. Mechanotransduction events are driven by the difference between the displacements in the two spaces, corresponding to the force acting on integrins. The equation for the displacement consists of two terms: one proportional to the radius that is the same in the intracellular and extracellular spaces (the monodomain term) and one that is proportional to a modified Bessel function that is responsible for mechanotransduction (the bidomain term). The model predicts that mechanotransduction occurs within a few length constants of the colony’s edge, and an expression for the length constant contains the intracellular and extracellular shear moduli and the spring constant of the integrins coupling the two spaces. The model predictions are qualitatively consistent with experiments on human embryonic stem cell colonies, in which differentiation is localized near the edge.

Keywords: cell colony, integrin, mechanical bidomain model, stem cell, stress-strain, traction force

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Authors: Masoud Sakhinia, Sajjad Ahmad

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Introduction: Though knowledge of the compositional makeup and structure of the limbal niche has progressed exponentially during the past decade, much is yet to be understood. Identifying the precise profile and role of the stromal makeup which spans the ocular surface may inform researchers of the most optimum conditions needed to effectively expand LESCs in vitro, whilst preserving their differentiation status and phenotype. Limbal fibroblasts, as opposed to corneal fibroblasts are thought to form an important component of the microenvironment where LESCs reside. Methods: The corneal stroma was tissue engineered in vitro using both limbal and corneal fibroblasts embedded within a tissue engineered 3D collagen matrix. The effect of these two different fibroblasts on LESCs and hTCEpi corneal epithelial cell line were then subsequently determined using phase contrast microscopy, histolological analysis and PCR for specific stem cell markers. The study aimed to develop an in vitro model which could be used to determine whether limbal, as opposed to corneal fibroblasts, maintained the stem cell phenotype of LESCs and hTCEpi cell line. Results: Tissue culture analysis was inconclusive and required further quantitative analysis for remarks on cell proliferation within the varying stroma. Histological analysis of the tissue-engineered cornea showed a comparable structure to that of the human cornea, though with limited epithelial stratification. PCR results for epithelial cell markers of cells cultured on limbal fibroblasts showed reduced expression of CK3, a negative marker for LESC’s, whilst also exhibiting a relatively low expression level of P63, a marker for undifferentiated LESCs. Conclusion: We have shown the potential for the construction of a tissue engineered human cornea using a 3D collagen matrix and described some preliminary results in the analysis of the effects of varying stroma consisting of limbal and corneal fibroblasts, respectively, on the proliferation of stem cell phenotype of primary LESCs and hTCEpi corneal epithelial cells. Although no definitive marker exists to conclusively illustrate the presence of LESCs, the combination of positive and negative stem cell markers in our study were inconclusive. Though it is less traslational to the human corneal model, the use of conditioned medium from that of limbal and corneal fibroblasts may provide a more simple avenue. Moreover, combinations of extracellular matrices could be used as a surrogate in these culture models.

Keywords: cornea, Limbal Stem Cells, tissue engineering, PCR

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Authors: Lu-Chen Yeh‚ Jui-Ming Yeh

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In this manuscript, we produced neat electrospun poly(o-methoxyaniline) (POMA) fibers and utilized it for applying the growth of neural stem cells. The transparency and morphology of as-prepared POMA fibers were characterized by UV-visible spectroscopy and scanning electron microscopy, respectively. It was found to have no adverse effects on the long-term proliferation of the neural stem cells (NSCs), retained the ability to self-renew, and exhibit multi-potentiality. Results of immunofluorescence staining studies confirmed that POMA electrospun fibers could provide a great environment for NSCs and enhance its differentiation.

Keywords: electrospun, polyaniline, neural stem cell, differentiation

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Authors: Arzu Karahan, Loriano Ballarin, Baruch Rinkevich

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Marine invertebrates, the highly diverse phyla of multicellular organisms, represent phenomena that are either not found or highly restricted in the vertebrates. These include phenomena like budding, fission, a fusion of ramets, and high regeneration power, such as the ability to create whole new organisms from either tiny parental fragment, many of which are controlled by totipotent, pluripotent, and multipotent stem cells. Thus, there is very much that can be learned from these organisms on the practical and evolutionary levels, further resembling Darwin's words, “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change”. The ‘stem cell’ notion highlights a cell that has the ability to continuously divide and differentiate into various progenitors and daughter cells. In vertebrates, adult stem cells are rare cells defined as lineage-restricted (multipotent at best) with tissue or organ-specific activities that are located in defined niches and further regulate the machinery of homeostasis, repair, and regeneration. They are usually categorized by their morphology, tissue of origin, plasticity, and potency. The above description not always holds when comparing the vertebrates with marine invertebrates’ stem cells that display wider ranges of plasticity and diversity at the taxonomic and the cellular levels. While marine/aquatic invertebrates stem cells (MISC) have recently raised more scientific interest, the know-how is still behind the attraction they deserve. MISC, not only are highly potent but, in many cases, are abundant (e.g., 1/3 of the entire animal cells), do not locate in permanent niches, participates in delayed-aging and whole-body regeneration phenomena, the knowledge of which can be clinically relevant. Moreover, they have massive hidden potential for the discovery of new bioactive molecules that can be used for human health (antitumor, antimicrobial) and biotechnology. The MARISTEM COST action (Stem Cells of Marine/Aquatic Invertebrates: From Basic Research to Innovative Applications) aims to connect the European fragmented MISC community. Under this scientific umbrella, the action conceptualizes the idea for adult stem cells that do not share many properties with the vertebrates’ stem cells, organizes meetings, summer schools, and workshops, stimulating young researchers, supplying technical and adviser support via short-term scientific studies, making new bridges between the MISC community and biomedical disciplines.

Keywords: aquatic/marine invertebrates, adult stem cell, regeneration, cell cultures, bioactive molecules

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Authors: Nishanth Venugopal Menon, Chuah Yon Jin, Samantha Phey, Wu Yingnan, Zhang Ying, Vincent Chan, Kang Yuejun

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Cell Migration is a vital phenomenon that the cells undergo in various physiological processes like wound healing, disease progression, embryogenesis, etc. Cell migration depends primarily on the chemical and physical cues available in the cellular environment. The chemical cue involves the chemokines secreted and gradients generated in the environment while physical cues indicate the impact of matrix properties like nanotopography and stiffness on the cells. Mesenchymal Stem Cells (MSCs) have been shown to have a role wound healing in vivo and its migration to the site of the wound has been shown to have a therapeutic effect. In the field of stem cell based tissue regeneration of bones and cartilage, one approach has been to introduce scaffold laden with MSCs into the site of injury to enable tissue regeneration. In this work, we have studied the combinatorial impact of the substrate physical properties on MSC migration. A microfluidic in vitro model was created to perform the migration studies. The microfluidic model used is a three compartment device consisting of two cell seeding compartments and one migration compartment. Four different PDMS substrates with varying substrate roughness, stiffness and hydrophobicity were created. Its surface roughness and stiffness was measured using Atomic Force Microscopy (AFM) while its hydrphobicity was measured from the water contact angle using an optical tensiometer. These PDMS substrates are sealed to the microfluidic chip following which the MSCs are seeded and the cell migration is studied over the period of a week. Cell migration was quantified using fluorescence imaging of the cytoskeleton (F-actin) to find out the area covered by the cells inside the migration compartment. The impact of adhesion proteins on cell migration was also quantified using a real-time polymerase chain reaction (qRT PCR). These results suggested that the optimal substrate for cell migration would be one with an intermediate level of roughness, stiffness and hydrophobicity. A higher or lower value of these properties affected cell migration negatively. These observations have helped us in understanding that different substrate properties need to be considered in tandem, especially while designing scaffolds for tissue regeneration as cell migration is normally impacted by the combinatorial impact of the matrix. These observations may lead us to scaffold optimization in future tissue regeneration applications.

Keywords: cell migration, microfluidics, in vitro model, stem cell migration, scaffold, substrate properties

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Authors: Urvi Panwar, Kanchan Mishra, Kanjaksha Ghosh, ShankerLal Kothari

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Background: Day-by-day the increasing prevalence of neurodegenerative diseases have become a global issue to manage them by medical sciences. The adult neural stem cells are rare and require an invasive and painful procedure to obtain it from central nervous system. Mesenchymal stem cell (MSCs) therapies have shown remarkable application in treatment of various cell injuries and cell loss. MSCs can be derived from various sources like adult tissues, human bone marrow, umbilical cord blood and cord tissue. MSCs have similar proliferation and differentiation capability, but the human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are proved to be more beneficial with respect to cell procurement, differentiation to other cells, preservation, and transplantation. Material and method: Human umbilical cord is easily obtainable and non-controversial comparative to bone marrow and other adult tissues. The umbilical cord can be collected after delivery of baby, and its tissue can be cultured using explant culture method. Cell culture medium such as DMEMF12+10% FBS and DMEMF12+Neural growth factors (bFGF, human noggin, B27) with antibiotics (Streptomycin/Gentamycin) were used to culture and differentiate mesenchymal stem cells into neural cells, respectively. The characterisations of MSCs were done with Flow Cytometer for surface markers CD90, CD73 and CD105 and colony forming unit assay. The differentiated various neural cells will be characterised by fluorescence markers for neurons, astrocytes, and oligodendrocytes; quantitative PCR for genes Nestin and NeuroD1 and Western blotting technique for gap43 protein. Result and discussion: The high quality and number of MSCs were isolated from human umbilical cord via explant culture method. The obtained MSCs were differentiated into neural cells like neurons, astrocytes and oligodendrocytes. The differentiated neural cells can be used to treat neural injuries and neural cell loss by delivering cells by non-invasive administration via cerebrospinal fluid (CSF) or blood. Moreover, the MSCs can also be directly delivered to different injured sites where they differentiate into neural cells. Therefore, human umbilical cord is demonstrated to be an inexpensive and easily available source for MSCs. Moreover, the hUCMSCs can be a potential source for neural cell therapies and neural cell regeneration for neural cell injuries and neural cell loss. This new way of research will be helpful to treat and manage neural cell damages and neurodegenerative diseases like Alzheimer and Parkinson. Still the study has a long way to go but it is a promising approach for many neural disorders for which at present no satisfactory management is available.

Keywords: bone marrow, cell therapy, explant culture method, flow cytometer, human umbilical cord, mesenchymal stem cells, neurodegenerative diseases, neuroprotective, regeneration

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Authors: Geoffrey A. Wright

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Over the past year we have developed and implemented a blended STEM curriculum based on ROV (Remotely Operated Vehicle) underwater technology with over 300 students in grades 2–9. This paper presents an overview of the curriculum, what we have learned from the development and implementation, with suggestions of how to build a similar statewide ROV program, and how we will continue and enhance the effort this next year with more than 300 additional students. The benefits of the program are the application and blending of STEM principles using inquiry based instruction, where students have shown to increase in STEM self-efficacy and interest.

Keywords: STEM, technology, engineering, ROV

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Authors: Ali A. Alshatwi, Vaiyapuri S. Periasamy, Jegan Athinarayanan

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Engineered nanoparticles’ usage rapidly increased in various applications in the last decade due to their unusual properties. However, there is an ever increasing concern to understand their toxicological effect in human health. Particularly, metal and metal oxide nanoparticles have been used in various sectors including biomedical, food and agriculture. But their impact on human health is yet to be fully understood. In this present investigation, we assessed the toxic effect of engineered nanoparticles (ENPs) including Ag, MgO and Co3O4 nanoparticles (NPs) on human mesenchymal stem cells (hMSC) adopting cell viability and cellular morphological changes as tools The results suggested that silver NPs are more toxic than MgO and Co3O4NPs. The ENPs induced cytotoxicity and nuclear morphological changes in hMSC depending on dose. The cell viability decreases with increase in concentration of ENPs. The cellular morphology studies revealed that ENPs damaged the cells. These preliminary findings have implications for the use of these nanoparticles in food industry with systematic regulations.

Keywords: cobalt oxide, human mesenchymal stem cells, MgO, silver

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Authors: Nunthawan Nowwarote, Waleerat Sukarawan, Prasit Pavasant, Thanaphum Osathanon

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Interleukin 6 (IL-6) is a multifunctional cytokine, regulating various biological responses in several tissues. A Recent study shows that IL-6 plays a role in stemness maintenance in stem cells isolated from human exfoliated deciduous teeth (SHEDs). However, the role of IL-6 on cell differentiation in SHEDs remains unknown. The present study investigated the effect of IL-6 on SHEDs differentiation. Cells were isolated from dental pulp tissues of human deciduous teeth. Flow cytometry was used to determined mesenchymal stem cell marker expression, and the multipotential differentiation (osteogenic, adipogenic and neurogenic lineage ) was also determined. The mRNA was determined using real-time quantitative polymerase chain reaction, and the phenotypes were confirmed by chemical and immunofluorescence staining. Results demonstrated that SHEDs expressed CD44, CD73, CD90, CD105 but not CD45. Further, the up-regulation of osteogenic, adipogenic and neurogenic marker genes was observed upon maintaining cells in osteogenic, adipogenic and neurogenic induction medium, respectively. The addition of IL-6 induced osteogenic by up-regulated osteogenic marker gene also increased in vitro mineralization. Under neurogenic medium supplement with IL-6, up-regulated neurogenic marker. Whereas, an addition of IL-6 attenuated adipogenic differentiation by SHEDs. In conclusion, this evidence implies that IL-6 may participate in cells differentiation ability of SHEDs.

Keywords: SHEDs, IL-6, cell differentiations, dental pulp

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Authors: W. Baumgartner, M. Welti, N. Hild, S. C. Hess, W. J. Stark, G. Meier Bürgisser, P. Giovanoli, J. Buschmann

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Perfusion bioreactors are used to solve problems in tissue engineering in terms of sufficient nutrient and oxygen supply. Such problems especially occur in critical size grafts because vascularization is often too slow after implantation ending up in necrotic cores. Biominerizable and biocompatible nanocomposite materials are attractive and suitable scaffold materials for bone tissue engineering because they offer mineral components in organic carriers – mimicking natural bone tissue. In addition, human adipose derived stem cells (ASCs) can potentially be used to increase bone healing as they are capable of differentiating towards osteoblasts or endothelial cells among others. In the present study, electrospun nanocomposite disks of poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/a-CaP) were seeded with human ASCs and eight disks were stacked in a bioreactor running with normal culture medium (no differentiation supplements). Under continuous perfusion and uniaxial cyclic compression, load-displacement curves as a function of time were assessed. Stiffness and energy dissipation were recorded. Moreover, stem cell densities in the layers of the piled scaffold were determined as well as their morphologies and differentiation status (endothelial cell differentiation, chondrogenesis and osteogenesis). While the stiffness of the cell free constructs increased over time caused by the transformation of the a-CaP nanoparticles into flake-like apatite, ASC-seeded constructs showed a constant stiffness. Stem cell density gradients were histologically determined with a linear increase in the flow direction from the bottom to the top of the 3.5 mm high pile (r2 > 0.95). Cell morphology was influenced by the flow rate, with stem cells getting more roundish at higher flow rates. Less than 1 % osteogenesis was found upon osteopontin immunostaining at the end of the experiment (9 days), while no endothelial cell differentiation and no chondrogenesis was triggered under these conditions. All ASCs had mainly remained in their original pluripotent status within this time frame. In summary, we have fabricated a critical size bone graft based on a biominerizable bone-biomimetic nanocomposite with preserved stiffness when seeded with human ASCs. The special feature of this bone graft was that ASC densities inside the piled construct varied with a linear gradient, which is a good starting point for tissue engineering interfaces such as bone-cartilage where the bone tissue is cell rich while the cartilage exhibits low cell densities. As such, this tissue-engineered graft may act as a bone-cartilage interface after the corresponding differentiation of the ASCs.

Keywords: bioreactor, bone, cartilage, nanocomposite, stem cell gradient

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Authors: Fui Ping Lim, Wen Choong Chua, Toan Thang Phan

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Aim: This study investigates the roles of Cord Lining Stem Cells (CLSCs) as potential therapeutic agents for diabetic wounds. Method: 20 genetically diabetic db/db mice were randomly assigned to two arms; (i) control group received placebo treatment (sham media or cells delivery material), and (ii) active comparator received CLSCs. Two full-thickness wounds, each sized 10mm X 10mm were created, one on each side of the midline on the back of the mice. Digital pictures were taken on day 1, 3, 7, 10, 14, 17, 21, 24, 28. Wound areas were analyzed with ImageJ TM software and calculated as percentage of the original wound. Time to closure was defined as the day the wound bed was completely epithelized and filled with new tissues. Results: The CLSCs-treated wounds, showed a significant increase in the percentage of wound closure and achieved 100% closure of the wound sooner than the control group by an average of 3.7 days. The mice treated with CLSCs have a shorter wound closure time (mean closure day: 19.8 days) as compared to the control group (mean closure day: 23.5 days). Conclusion: Our preliminary findings inferred that CLSCs treated wound achieved higher percentage of wound closure within a shorter duration of time.

Keywords: cord lining stem cell, diabetic wound, stem cell, wound

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Authors: Roxane A. Legaie, Kjiana E. Schwab, Caroline E. Gargett

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Studies looking at the changes in gene expression from RNAseq data often make use of linear models. It is also common practice to focus on a subset of data for a comparison of interest, leaving aside the samples not involved in this particular comparison. This work shows the importance of including all observations in the modeling process to better estimate variance parameters, even when the samples included are not directly used in the comparison under test. The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. The mesenchymal stem cells (MSCs) present in the endometrium are likely responsible for this remarkable regenerative capacity. However recent studies suggest that MSCs also plays a role in the pathogenesis of endometriosis, one of the most common medical conditions affecting the lower abdomen in women in which the endometrial tissue grows outside the womb. In this study we compared gene expression profiles between MSCs and non-stem cell counterparts (‘non-MSC’) obtained from women with (‘E’) or without (‘noE’) endometriosis from RNAseq. Raw read counts were used for differential expression analysis using a linear model with the limma-voom R package, including either all samples in the study or only the samples belonging to the subset of interest (e.g. for the comparison ‘E vs noE in MSC cells’, including only MSC samples from E and noE patients but not the non-MSC ones). Using the full dataset we identified about 100 differentially expressed (DE) genes between E and noE samples in MSC samples (adj.p-val < 0.05 and |logFC|>1) while only 9 DE genes were identified when using only the subset of data (MSC samples only). Important genes known to be involved in endometriosis such as KLF9 and RND3 were missed in the latter case. When looking at the MSC vs non-MSC cells comparison, the linear model including all samples identified 260 genes for noE samples (including the stem cell marker SUSD2) while the subset analysis did not identify any DE genes. When looking at E samples, 12 genes were identified with the first approach and only 1 with the subset approach. Although the stem cell marker RGS5 was found in both cases, the subset test missed important genes involved in stem cell differentiation such as NOTCH3 and other potentially related genes to be used for further investigation and pathway analysis.

Keywords: differential expression, endometriosis, linear model, RNAseq

Procedia PDF Downloads 402

Authors: Gileade P. Freitas, Helena B. Lopes, Alann T. P. Souza, Paula G. F. P. Oliveira, Adriana L. G. Almeida, Paulo G. Coelho, Marcio M. Beloti, Adalberto L. Rosa

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Bone tissue presents great capacity to regenerate when injured by trauma, infectious processes, or neoplasia. However, the extent of injury may exceed the inherent tissue regeneration capability demanding some kind of additional intervention. In this scenario, cell therapy has emerged as a promising alternative to treat challenging bone defects. This study aimed at evaluating the effect of local injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) on bone regeneration of rat calvaria defects. BM-MSCs and AT-MSCs were isolated and characterized by expression of surface markers; cell viability was evaluated after injection through a 21G needle. Defects of 5 mm in diameter were created in calvaria and after two weeks a single injection of BM-MSCs, AT-MSCs or vehicle-PBS without cells (Control) was carried out. Cells were tracked by bioluminescence and at 4 weeks post-injection bone formation was evaluated by micro-computed tomography (μCT) and histology, nanoindentation, and through gene expression of bone remodeling markers. The data were evaluated by one-way analysis of variance (p≤0.05). BM-MSCs and AT-MSCs presented characteristics of mesenchymal stem cells, kept viability after passing through a 21G needle and remained in the defects until day 14. In general, injection of both BM-MSCs and AT-MSCs resulted in higher bone formation compared to Control. Additionally, this bone tissue displayed elastic modulus and hardness similar to the pristine calvaria bone. The expression of all evaluated genes involved in bone formation was upregulated in bone tissue formed by BM-MSCs compared to AT-MSCs while genes involved in bone resorption were upregulated in AT-MSCs-formed bone. We show that cell therapy based on the local injection of BM-MSCs or AT-MSCs is effective in delivering viable cells that displayed local engraftment and induced a significant improvement in bone healing. Despite differences in the molecular cues observed between BM-MSCs and AT-MSCs, both cells were capable of forming bone tissue at comparable amounts and properties. These findings may drive cell therapy approaches toward the complete bone regeneration of challenging sites.

Keywords: cell therapy, mesenchymal stem cells, bone repair, cell culture

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Authors: Abdulwali H. Aldahmash, Naem M. Alamri

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The purpose of this study was to analyze Saudi Arabian science and mathematics teachers’ attitudes toward integrating STEM in teaching before and after they participated in a professional development workshop focused on STEM integration in a specific middle school science and mathematics unit. The participants were 48 Saudi Arabian science and mathematics teachers who participated in a three-day workshop held in Riyadh, Saudi Arabia. The research method was a pretest-posttest group design. The primary data source was the instrument for teachers' attitudes toward teaching integrated STEM. The results indicate that Saudi Arabian science and mathematics teachers’ perceptions of difficulties decreased due to their participation in the professional development workshop on integrated STEM. Meanwhile, the teachers' self-efficacy improved following their participation in the STEM professional development (PD) workshop. However, no perceived effect was found for the teachers' perceptions of the relevance of or their anxiety about or enjoyment of integrated STEM teaching due to their participation in the three-day PD workshop.

Keywords: STEM integration, attitude toward STEM, STEM workshop, professional development

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Authors: Mariyah Poonawala, Selvan Ravindran, Anuradha Vaidya

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Despite much progress in understanding the regulatory factors and cytokines that support the maturation of the various cell lineages of the hematopoietic system, factors that govern the self-renewal and proliferation of hematopoietic stem cells (HSCs) is still a grey area of research. Hematopoietic stem cell transplantation (HSCT) has evolved over the years and gained tremendous importance in the treatment of both malignant and non-malignant diseases. However, factors such as graft rejection and multiple organ failure have challenged HSCT from time to time, underscoring the urgent need for development of milder processes for successful hematopoietic transplantation. An emerging concept in the field of stem cell biology states that the interactions between the bone-marrow micro-environment and the hematopoietic stem and progenitor cells is essential for regulation, maintenance, commitment and proliferation of stem cells. Understanding the role of mesenchymal stromal cells in modulating the functionality of HSCs is, therefore, an important area of research. Trans-resveratrol has been extensively studied for its various properties to combat and prevent cancer, diabetes and cardiovascular diseases etc. The aim of the present study was to understand the effect of trans-resveratrol on HSCs using single and co-culture systems. We have used KG1a cells since it is a well accepted hematopoietic stem cell model system. Our preliminary experiments showed that low concentrations of trans-resveratrol stimulated the HSCs to undergo proliferation whereas high concentrations of trans-resveratrol did not stimulate the cells to proliferate. We used a murine fibroblast cell line, M210B4, as a stromal feeder layer. On culturing the KG1a cells with M210B4 cells, we observed that the stimulatory as well as inhibitory effects of trans-resveratrol at low and high concentrations respectively, were enhanced. Our further experiments showed that low concentration of trans-resveratrol reduced the generation of reactive oxygen species (ROS) and nitric oxide (NO) whereas high concentrations increased the oxidative stress in KG1a cells. We speculated that perhaps the oxidative stress was imposing inhibitory effects at high concentration and the same was confirmed by performing an apoptotic assay. Furthermore, cell cycle analysis and growth kinetic experiments provided evidence that low concentration of trans-resveratrol reduced the doubling time of the cells. Our hypothesis is that perhaps at low concentration of trans-resveratrol the cells get pushed into the G0/G1 phase and re-enter the cell cycle resulting in their proliferation, whereas at high concentration the cells are perhaps arrested at G2/M phase or at cytokinesis and therefore undergo apoptosis. Liquid Chromatography-Quantitative-Time of Flight–Mass Spectroscopy (LC-Q-TOF MS) analyses indicated the presence of trans-resveratrol and its metabolite(s) in the supernatant of the co-cultured cells incubated with high concentration of trans-resveratrol. We conjecture that perhaps the metabolites of trans-resveratrol are responsible for the apoptosis observed at the high concentration. Our findings may shed light on the unsolved problems in the in vitro expansion of stem cells and may have implications in the ex vivo manipulation of HSCs for therapeutic purposes.

Keywords: co-culture system, hematopoietic micro-environment, KG1a cell line, M210B4 cell line, trans-resveratrol

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Authors: Elham Vojoudi, Marzieh Mehrafza, Ahmad Hosseini, Azadeh Raofi, Maryam Najafi

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Premature ovarian failure (POF) has become one of the main causes of infertility in women of childbearing age and the incidence of this disorder is increasing year by year. In these patients, poor ovarian response (POR) to gonadotropins reflects a diminished ovarian reserve (DOR) that gives place to few follicles despite aggressive stimulation. Up to now, egg donation is the only way to resolve infertility problems in POF patients. Therefore, some novel aspects such as activating (Akt signaling pathway) and inhibiting (Hippo-signaling) elements have been identified as IVA procedure that promotes primordial follicle activation. In this study, we used the newly developed technique (combination of in vitro activation of dormant follicles (IVA) and stem cell therapy) to promote ovarian follicle growth much more efficiently than the natural, in vivo process for women with POF. Transplantation of Warton Jelly-MSCs to the ovaries of POF patients rescued overall ovarian function. Participants (10 patients) were followed up monthly for a period of six months by hormonal (AMH, FSH, LH and E2), clinical (resuming menstruation), and US (folliculometry) outcomes after a laparoscopic operation. In summary, IVA/WJ-MSC transplantation may provide an effective treatment for POF.

Keywords: POF, in vitro activation, stem cell therapy, infertility

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Authors: Igor Sukhotnik

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Objectives: Notch signaling is thought to act to drive cell versification in the lining of the small intestine. When Notch signaling is blocked, proliferation ceases, and epithelial cells become secretory. The purpose of the present study was to evaluate the role of Notch signaling pathway in stem cell differentiation in a rat model of intestinal ischemia-reperfusion (IR). Methods: Male Sprague-Dawley rats were randomly divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were killed 24 or 48 h later, respectively; IR-24 and IR-48 rats underwent occlusion of SMA and portal vein for 30 min followed by 24 or 48 h of reperfusion, respectively. Notch-related gene and protein expression were determined using Real Time PCR, Western blotting and immunohistochemistry. Wax histology and immunohistochemistry was used to determine cell differentiation toward absorptive (enterocytes) or secretory progenitors (goblet cells, enteroendocrine cells or Paneth cells). Results: IR-48 rats exhibited a significant decrease in Notch-1 protein expression (Western blot) that was coincided with a significant decrease in the number of Notch-1 positive cells (immunohistochemistry) in jejunum and ileum as well as Hes-1 positive cells in jejunum and ileum compared to Sham-48 rats. A significant down-regulation of Notch signaling related genes and proteins in IR animals was accompanied by a significant increase in the number of goblet and Paneth cells and decreased number of absorptive cells compared to control rats. Conclusions: Forty-eight hours following intestinal IR in rats, inhibited Notch signaling pathway was accompanied by intestinal stem cells differentiation toward secretory progenitors.

Keywords: Intestine, notch, ischemia-reperfusion, cell differentiation, secretory

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Authors: Alieh Farshbaf, Tayyeb Bahrami

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Introduction: Cc-chemokine receptor-5 (CCR5) is known as a main co-receptor in human immunodeficiency virus type-1 (HIV-1) infection. Many studies showed 32bp deletion (Δ32) in CCR5 gene, provide natural resistance to HIV-1 infection in homozygous individuals. Inducing the resistance mechanism by CCR5 in HIV-1 infected patients eliminated many problems of highly-active-anti retroviral therapy (HAART) drugs like as low safety, side-effects and virus rebounding from latent reservoirs. New treatments solved some restrictions that are based on gene modification and cell therapy. Literature review: The stories of the “Berlin and Boston patients” showed autologous hematopoietic stem cells transplantation (HSCT) could provide effective cure of HIV-1 infected patients. Furthermore, gene modification by zinc finger nuclease (ZFN) demonstrated another successful result again. Despite the other studies for gene therapy by ∆32 genotype, there is another mutation -CCR5 ∆32/m303- that provides HIV-1 resistant. It is a heterozygote genotype for ∆32 and T→A point mutation at nucleotide 303. These results approved the key role of CCR5 gene. Conclusion: Recent studies showed immune gene therapy and cell therapy could provide effective cure for refractory disease like as HIV. Eradication of HIV-1 from immune system was not observed by HAART, because of reloading virus genome from latent reservoirs after stopping them. It is showed that CCR5 could induce natural resistant to HIV-1 infection by the new approaches based on stem cell transplantation and gene modifying.

Keywords: CCR5, HIV-1, stem cell, immune gene therapy, gene modification

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