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The microRNAs (miRNAs) are small, non-coding RNAs that modulate protein expression by interfering with target mRNA translation or stability. miRNAs play crucial roles in various functions such as cellular, developmental, and physiological processes. The spatial expression patterns of miRNAs are very essential for identifying their functions. The expressions of miR-302 and miR-367 are critical in maintaining stemness of pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) but their functions in early development are not fully elucidated. So, we used Locked Nucleic Acid (LNA) probes to perform in situ hybridization and confirmed the temporal and spatial distribution patterns during early chick development. As a result, we found that miR-302 and miR-367 were expressed in various tissues such as primitive steak, neural ectoderm, neural plate, neural fold, neural tube, notochord, and oral cavity. Specially, we confirmed that miR-302 and miR-367 were strongly expressed in neural folds in HH8 to HH10. miR-302 was expressed on dorsal part of the neural tube but miR-367 was expressed on lateral and ventral parts of the neural tube. And also we performed quantitative stem-loop real-time PCR to analyze global expression level of miR-302 and miR-367. miR-302 and miR-367 expression was sustained before Hamburger and Hamilton stage (HH) 14. Thus, the temporal and spatial expression patterns of miR-302 and miR-367 may provide us information of the role of these miRNAs on tissue formation during early chick development.
Ectoderm ; Embryonic Stem Cells ; In Situ Hybridization ; Induced Pluripotent Stem Cells ; MicroRNAs ; Mouth ; Neural Crest ; Neural Plate ; Neural Tube ; Notochord ; Physiological Processes ; Pluripotent Stem Cells ; Protein Biosynthesis ; Real-Time Polymerase Chain Reaction ; RNA, Untranslated

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Combining Autologous Peripheral Blood Mononuclear Cells with Fibroblast Growth Factor Therapy Along with Stringent Infection Control Leading to Successful Limb Salvage in Diabetic Patient with Chronic Renal Failure and Severe Toe Gangrene.

Hiroshi OSAWA ; Kouan ORII ; Hiroshi TERUNUMA ; Samuel J K ABRAHAM

International Journal of Stem Cells.2014;7(2):158-161. doi:10.15283/ijsc.2014.7.2.158

Peripheral arterial disease (PAD) is a common complication of Diabetes Mellitus (DM) and often culminates in amputation of the affected foot. Pseudomonas aeruginosa infections associated with PAD are difficult to treat due to their multi-drug resistance. Herein we report a 38 year old male who reported with DM, chronic kidney disease (CKD) and rest pain of the right second toe in October 2011. He underwent percutaneous transluminal angioplasty (PTA) which was unsuccessful. The gangrene of the toes worsened and amputation of the right second toe was done. Bacteriological examination showed presence of P. aeruginosa which during the course of antibiotic therapy became multi-drug resistant. Gangrene and abscess of the foot worsened and amputation of the right third toe was performed. Then autologous peripheral blood mononuclear cell (PBMNC) therapy was performed but as infection control could not still be achieved, the fourth toe was amputated. A protocol of foot bath using carbonic water, local usage of antibiotics (Polymyxin-B), and basic fibroblast growth factor (b-FGF) spray was then employed after which the infection could be controlled and improvement in vascularity of the right foot could be observed in angiography. This combined approach after proper validation could be considered for similar cases.
Abscess ; Amputation ; Angiography ; Angioplasty ; Anti-Bacterial Agents ; Baths ; Carbon ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus ; Drug Resistance, Multiple ; Fibroblast Growth Factor 2 ; Fibroblast Growth Factors* ; Foot ; Gangrene* ; Humans ; Infection Control* ; Kidney Failure, Chronic* ; Limb Salvage* ; Male ; Peripheral Arterial Disease ; Pseudomonas aeruginosa ; Renal Insufficiency, Chronic ; Toes* ; Water

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Exogenous c-Myc Blocks Differentiation and Improves Expansion of Human Erythroblasts In vitro.

Cristopher GEILER ; Inez ANDRADE ; Daniel GREENWALD

International Journal of Stem Cells.2014;7(2):153-157. doi:10.15283/ijsc.2014.7.2.153

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the blood supply for transfusion treatment. The production of red blood cells from hematopoietic stem cells in the laboratory is possible but the mass production of red blood cells to the level present in a blood transfusion unit is currently not possible. The proliferation capacity of the immature red blood cell will need to be increased to enable mass production. This work focused on the hypothesis that exogenous c-Myc can delay the differentiation process of highly proliferative immature erythroblasts, and increase the proliferation capacity of erythroblast cell cultures. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis from stem cells without gene transfection with the eventual goal of producing blood for transfusion treatment in a manner that could be easily translated into clinical medicine. METHODS: The hematopoietic stem cell containing mononuclear cell fraction of venous blood samples was cultured in a liquid media containing erythroblasts growth factors with and without exogenous c-Myc combined with a cell-penetrating peptide. The cells were maintained in the liquid culture media for 23 days. Viable cells were counted and analyzed with flow cytometry. RESULTS: Our results show a 4 fold increase in expansion of the erythroblasts grown in the c-Myc containing growth media compared to the control. Eighty percent of these cells retained the CD117 surface receptor, indicating immature cells. CONCLUSION: Exogenous c-Myc blocks the differentiation and improves in vitro expansion of human erythroblasts.
Adult Stem Cells ; Blood Transfusion ; Cell Culture Techniques ; Clinical Medicine ; Culture Media ; Erythroblasts* ; Erythrocytes ; Erythropoiesis ; Flow Cytometry ; Hematopoietic Stem Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Proto-Oncogene Proteins c-myc ; Stem Cells ; Transfection

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Growth Properties and Pluripotency Marker Expression of Spontaneously Formed Three-dimensional Aggregates of Human Adipose-derived Stem Cells.

Ance BOGDANOVA-JATNIECE ; Uldis BERZINS ; Tatjana KOZLOVSKA

International Journal of Stem Cells.2014;7(2):143-152. doi:10.15283/ijsc.2014.7.2.143

BACKGROUND AND OBJECTIVES: Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor. METHODS AND RESULTS: Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected. CONCLUSIONS: Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.
Alkaline Phosphatase ; Glass ; Humans ; Mesenchymal Stromal Cells ; Plastics ; Stem Cells* ; Tissue Donors

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Characterization of Mesenchymal Stem Cells Derived from Rat Bone Marrow and Adipose Tissue: A Comparative Study.

Ahmed LOTFY ; Mohamed SALAMA ; Faten ZAHRAN ; Elena JONES ; Ahmed BADAWY ; Mohamed SOBH

International Journal of Stem Cells.2014;7(2):135-142. doi:10.15283/ijsc.2014.7.2.135

BACKGROUND AND OBJECTIVES: Stem cell technology offers a new hope for many chronic disorders patients. The types of stem cells are different with many differences existing between each type. Mesenchymal stem cells (MSCs) represent one type of adult stem cells that can be easily isolated, then re-transplanted to the patients. This offers potential for their future application in treating many disorders without fear of rejection possibility. MSCs can be isolated from different sources e.g. bone marrow (BMSCs) and adipose tissue (ADSCs). In the present study we compared BMSCs and ADSCs isolated from Sprague-Dawley rats. METHODS AND RESULTS: For this comparison, immunophenotyping, the analysis of growth rates, proliferation by colony forming unit-fibroblast assay, population doubling time, and trilineage differentiation assays were performed for both BMSCs and ADSCs. The findings revealed that despite no difference in immunphenotypic character between BMSC and ADSC, a better proliferative capacity was observed for ADSCs which would advocate their better use in regenerative applications. On the other hand, BMSCs showed more potential for osteogenic and chondrogenic differentiation. CONCLUSIONS: Our study showed that, despite many similarities between both types of cells, there are differences existing which can offer assistance on choosing type of cell to be used in specific diseases. Although ADSCs seem more promising for regenerative application generally, BMSCs may represent a better choice for treating bone disorders.
Adipose Tissue* ; Adult Stem Cells ; Animals ; Bone Marrow* ; Hand ; Hope ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells* ; Rats* ; Rats, Sprague-Dawley ; Stem Cells

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Mesengenic Differentiation: Comparison of Human and Rat Bone Marrow Mesenchymal Stem Cells.

Arianna SCUTERI ; Elisabetta DONZELLI ; Dana FOUDAH ; Cristina CALDARA ; Juliana REDONDO ; Giovanna D'AMICO ; Giovanni TREDICI ; Mariarosaria MILOSO

International Journal of Stem Cells.2014;7(2):127-134. doi:10.15283/ijsc.2014.7.2.127

BACKGROUND AND OBJECTIVES: Cellular therapies using Mesenchymal Stem Cells (MSCs) represent a promising approach for the treatment of degenerative diseases, in particular for mesengenic tissue regeneration. However, before the approval of clinical trials in humans, in vitro studies must be performed aimed at investigating MSCs' biology and the mechanisms regulating their proliferation and differentiation abilities. Besides studies on human MSCs (hMSCs), MSCs derived from rodents have been the most used cellular type for in vitro studies. Nevertheless, the transfer of the results obtained using animal MSCs to hMSCs has been hindered by the limited knowledge regarding the similarities existing between cells of different origins. Aim of this paper is to highlight similarities and differences and to clarify the sometimes reported different results obtained using these cells. METHODS AND RESULTS: We compare the differentiation ability into mesengenic lineages of rat and human MSCs cultured in their standard conditions. Our results describe in which way the source from which MSCs are derived affects their differentiation potential, depending on the mesengenic lineage considered. For osteogenic and chondrogenic lineages, the main difference between human and rat MSCs is represented by differentiation time, while for adipogenesis hMSCs have a greater differentiation potential. CONCLUSIONS: These results on the one hand suggest to carefully evaluate the transfer of results obtained with animal MSCs, on the other hand they offer a clue to better apply MSCs into clinical practice.
Adipogenesis ; Animals ; Biology ; Bone Marrow* ; Hand ; Humans ; Mesenchymal Stromal Cells* ; Rats* ; Regeneration ; Rodentia

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Comparison of Mesenchymal Stem Cell Markers in Multiple Human Adult Stem Cells.

Masoud MALEKI ; Farideh GHANBARVAND ; Mohammad Reza BEHVARZ ; Mehri EJTEMAEI ; Elham GHADIRKHOMI

International Journal of Stem Cells.2014;7(2):118-126. doi:10.15283/ijsc.2014.7.2.118

OBJECTIVES: Mesenchymal stem cells (MSCs) are adult stem cells which identified by adherence to plastic, expression of cell surface markers including CD44, CD90, CD105, CD106, CD166, and Stro-1, lack of the expression of hematopoietic markers, no immunogenic effect and replacement of damaged tissues. These properties led to development of progressive methods to isolation and characterization of MSCs from various sources for therapeutic applications in regenerative medicine. METHODS: We isolated MSC-like cells from testis biopsies, ovary, hair follicle and umbilical cord Wharton's jelly and investigated the expression of specific cell surface antigens using flow cytometry in order to verify stemness properties of these cells. RESULTS: All four cell types adhered to plastic culture flask a few days after primary culture. All our cells positively expressed common MSC-specific cell surface markers. Moreover, our results revealed the expression of CD19and CD45 antigens in these cells. CONCLUSION: According to our results, high expression of CD44 in spermatogonial stem cells (SSCs), hair follicle stem cells (HFSCs),granulosa cells (GCs)and Wharton's jelly-MSCs (WJ-MSCs)may help them to maintain stemness properties. Furthermore, we suggest that CD105+SSCs, HFSCs and WJ-MSCs revealed the osteogenic potential of these cells. Moreover, high expression of CD90 in SSCs and HFSCs may associate to higher growth and differentiation potential of these cells. Further, the presence of CD19 on SSCs and GCs may help them to efficiency in response to transmembrane signals. Thus, these four types of MSCs may be useful in clinical applications and cell therapy.
Adult Stem Cells* ; Antigens, CD45 ; Antigens, Surface ; Biopsy ; Cell- and Tissue-Based Therapy ; Female ; Flow Cytometry ; Hair Follicle ; Humans ; Mesenchymal Stromal Cells* ; Ovary ; Plastics ; Regenerative Medicine ; Stem Cells ; Testis ; Umbilical Cord ; Wharton Jelly

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Transcriptional Profiles of Imprinted Genes in Human Embryonic Stem Cells During In vitro Differentiation.

Sang Wook PARK ; Hyo Sang DO ; Dongkyu KIM ; Ji Yun KO ; Sang Hun LEE ; Yong Mahn HAN

International Journal of Stem Cells.2014;7(2):108-117. doi:10.15283/ijsc.2014.7.2.108

BACKGROUND AND OBJECTIVES: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. METHODS AND RESULTS: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. CONCLUSIONS: Transcriptional expression of imprinted genes is regulated in a cell type-specific manner in hESCs during in vitro differentiation.
Alleles ; Embryonic Development ; Embryonic Stem Cells* ; Female ; Genomic Imprinting ; Human Development ; Humans ; Parents ; Pluripotent Stem Cells ; Pregnancy ; Wills

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