scholarly journals Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

2014 ◽  
Vol 22 (3) ◽  
pp. 218-227 ◽  
Author(s):  
Roberta Targa STRAMANDINOLI-ZANICOTTI ◽  
André Lopes CARVALHO ◽  
Carmen Lúcia Kuniyoshi REBELATTO ◽  
Laurindo Moacir SASSI ◽  
Maria Fernanda TORRES ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Animal Model ◽  
Mesenchymal Stem Cells ◽  
Basic Research ◽  
Large Animal Model ◽  
Large Animal ◽  
In Vitro Differentiation

scholarly journals Acceleration of Bone Regeneration in Critical-Size Defect Using BMP-9-Loaded nHA/ColI/MWCNTs Scaffolds Seeded with Bone Marrow Mesenchymal Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Ran Zhang ◽  
Xuewen Li ◽  
Yao Liu ◽  
Xiaobo Gao ◽  
Tong Zhu ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Composite Scaffold ◽  
Marrow Mesenchymal Stem Cells

Biocompatible scaffolding materials play an important role in bone tissue engineering. This study sought to develop and characterize a nano-hydroxyapatite (nHA)/collagen I (ColI)/multi-walled carbon nanotube (MWCNT) composite scaffold loaded with recombinant bone morphogenetic protein-9 (BMP-9) for bone tissue engineering by in vitro and in vivo experiments. The composite nHA/ColI/MWCNT scaffolds were fabricated at various concentrations of MWCNTs (0.5, 1, and 1.5% wt) by blending and freeze drying. The porosity, swelling rate, water absorption rate, mechanical properties, and biocompatibility of scaffolds were measured. After loading with BMP-9, bone marrow mesenchymal stem cells (BMMSCs) were seeded to evaluate their characteristics in vitro and in a critical sized defect in Sprague-Dawley rats in vivo. It was shown that the 1% MWCNT group was the most suitable for bone tissue engineering. Our results demonstrated that scaffolds loaded with BMP-9 promoted differentiation of BMMSCs into osteoblasts in vitro and induced more bone formation in vivo. To conclude, nHA/ColI/MWCNT scaffolds loaded with BMP-9 possess high biocompatibility and osteogenesis and are a good candidate for use in bone tissue engineering.

1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES

2016 ◽  
Vol 28 (2) ◽  
pp. 130
Author(s):  
R. Sper ◽  
S. Simpson ◽  
X. Zhang ◽  
B. Collins ◽  
J. Piedrahita
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Animal Model ◽  
Regenerative Medicine ◽  
Cancer Biology ◽  
Large Animal Model ◽  
Large Animal ◽  
Fetal Fibroblasts ◽  
Transgene Transmission

Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

275 IN VITRO DIFFERENTIATION OF PORCINE MESENCHYMAL STEM CELLS INTO NEURON-LIKE CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 217
Author(s):  
B. Mohana Kumar ◽  
H. J. Song ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. A. Ock ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Young Adult ◽  
Expression Pattern ◽  
Model Systems ◽  
Systematic Evaluation ◽  
In Vitro Differentiation ◽  
Different Origins

The unique self-renewal ability for multilineage differentiation makes mesenchymal stem cells (MSCs) a promising cell source for regenerative medicine. However, before adapting in clinical trials, there is a need for a systematic evaluation of the in vitro differentiation process and the functional identities of cells generated using animal model systems. We have previously reported that MSCs derived from porcine umbilical cord blood (pUCB), post-natal bone marrow, and young adult bone marrow with typical phenotypic properties differentiate in vitro into various cells types of mesodermal lineage (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells, in press). In the present study, we examined whether porcine MSCs (pMSCs) obtained from different origins, such as UCB, post-natal (immediately after birth) bone marrow, and young adult (6 months old) bone marrow are capable of neuronal differentiation in vitro. Subconfluent cultures of pMSCs (2–5 passages) were induced to become neuroectodermal cells following the protocol described earlier (Woodbury et al. 2000 J. Neurosci. Res. 61, 364–370), with minor modifications. After induction, 75 80% of cells exhibited substantial morphological changes including appearance of a more spherical shape, with multipolar and extending processes arranged into a network-like structure. Besides acquiring a neuronal phenotype, pMSCs derived from different origins displayed the expression pattern of characteristic neural-specific markers including early, intermediate, and late neural cell types. Differentiated cells expressed markers such as nestin, microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN), βIII-tubulin, neurofilament-M (NF-M), glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), and myelin basic protein (MBP) by immunofluorescence analysis. Expression pattern was further confirmed by analyzing selected neuronal transcripts by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Neurally differentiated pMSCs from different origins showed significantly (P < 0.05) higher relative abundance of nestin, βIII-tubulin, NF-M, and GFAP, compared to cells in control. In conclusion, the morphological observations and expression of neuronal-specific markers support the fact that pMSCs from different origins possess the ability to differentiate in vitro into neuron-like cells upon exposure to appropriate stimuli. Furthermore, these findings provide additional support for the emerging concept that the establishment of cell lines from pMSCs can have a pivotal role in standardizing cell replacement strategies.

Abstract WP145: Safety of Intra-Arterial Mesenchymal Stem Cells in a Large Animal Model of Stroke: A Dose Escalation Study

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Mitsuyoshi Watanabe ◽  
Karen E Bates ◽  
Luis Guada ◽  
Kevin Ramdas ◽  
Aisha Khan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Animal Model ◽  
Mesenchymal Stem Cells ◽  
Dose Escalation ◽  
Cerebral Blood Volume ◽  
Maximum Tolerated Dose ◽  
Large Animal Model ◽  
Large Animal ◽  
Vessel Occlusion ◽  
Dose Escalation Study

Background: Despite the efficacy of thrombectomy for large vessel occlusion acute ischemic stroke (AIS) , ~50% of patients have significant residual deficits. Pre-clinical data on intra-arterially (IA) administered mesenchymal stem cells (MSCs) in stroke are promising and this approach is attractive for clinical application. While there is a concern for micro-occlusion with IA delivery due to the large size of MSCs, a dose of 1 x 10 5 MSCs given 24-48 hr in a rodent reperfusion middle cerebral artery occlusion (rMCAo) model has been shown to be safe and effective. As per STAIR recommendations, we performed a dose-escalation (DE) study of IA-MSCs in a large animal stroke model. Methods: An endovascular canine rMCAo model using retractable platinum coil for 60-120 min was established. At 48 hr post-rMCAo, allogeniec canine MSCs were delivered using a 0.0165” microcatheter in the ipsilateral upper cervical internal carotid artery in escalating doses (based on proportion of rodent to canine total cerebral blood volume). Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at 15-30 d post-rMCAo and brains were harvested. Results: Female canines (n=13), age 12-36 months, weighing 22-26 kg received IA MSCs ranging from 5-80 x 10 6 (M). At doses of 5-40 M IA-MSCs no neurological worsening was observed. Serial NDS and stroke volume on MRI showed no increase post-IA-MSCs and actually showed progressive reduction. A higher numerical reduction was seen in the 10-40 M groups compared to 5 M. However, in the one canine receiving 80 M IA-MSCs, there was significant worsening of the MCA-area infarction and NDS due to microembolization at this higher dose. Gross examinations and histopathology of brain tissue were consistent with ischemia. The brain of a canine receiving 80 M cells showed differentially aged areas of necrosis supporting two ischemic events. Neuroblasts, doublecortin-positive cells, and neovascularization were observed in canine brains suggesting regenerative mechanisms. Conclusions: These data suggest that IA-MSCs are safe in a large animal model up to 40 M IA-MSCs and is the maximum tolerated dose in this DE study. Furthermore, our data suggests that up to 40 M IA-MSCs may be promising in exploring efficacy in AIS.

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