Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair.

Proteomic profiling of forskolin-induced differentiated BeWo cells: an in-vitro model of cytotrophoblast differentiation

Placental trophoblastic differentiation is characterized by the fusion of monolayer cytotrophoblasts into syncytiotrophoblasts. During this process of differentiation, several morphological and biochemical changes are known to occur, and this model has been employed to investigate the changes that occur at the gene and protein level during differentiation. Using the sensitive technique of proteomics [two-dimensional gel electrophoresis (2DGE)], changes in protein profile were evaluated in the control and forskolin-induced differentiated cells of trophoblastic choriocarcinoma BeWo cell line. Several proteins were differentially expressed in control and differentiated cells. Four major proteins were up-regulated as assessed by silver staining, and were further characterized as c-h-ras p 21 (phosphorylated), retinoblastoma susceptibility protein I and integrase interactor protein 1. These proteins are known to play an important role in growth arrest of cells, and thus may play a role in initiating the process of differentiation.

Patterned growth and differentiation of neural cells on polymer derived carbon substrates with micro/nano structures in vitro

The growth of neuroblastoma (N2a) and Schwann cells has been explored on polymer derived carbon substrates of varying micro and nanoscale geometries: resorcinol-formaldehyde (RE) gel derived carbon films and electrospun nanofibrous (similar to 200 nm diameter) mat and SU-8 (a negative photoresist) derived carbon micro-patterns. MTT assay and complementary lactate dehydrogenase (LDH) assay established cytocompatibility of RE derived carbon films and fibers over a period of 6 days in culture. The role of length scale of surface patterns in eliciting lineage-specific adaptive response along, across and on the interspacing between adjacent micropatterns (i.e., ``on'', ``across'' and ``off'') has been assayed. Textural features were found to affect 3',5'-cyclic AMP sodium salt-induced neurite outgrowth, over a wide range of length scales: from similar to 200 nm (carbon fibers) to similar to 60 mu m (carbon patterns). Despite their innate randomness, carbon nanofibers promoted preferential differentiation of N2a cells into neuronal lineage, similar to ordered micro-patterns. Our results, for the first time, conclusively demonstrate the potential of RE-gel and SU-8 derived carbon substrates as nerve tissue engineering platforms for guided proliferation and differentiation of neural cells in vitro. (C) 2013 Elsevier Ltd. All rights reserved.

A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

Background: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. Methods: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. Results: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. Conclusions: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

High concentrations of dexamethasone suppresses the proliferation but not the differentiation of further maturation of human osteoblast precursor in vitro: relevance to glucocorticoid-induced osteoporosis

Objective. The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). Methods. Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 µm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. Results. At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the 2 and 4, but not ß1, integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. Conclusions. A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

This paper is novel andreports on the in vitro establishment of 3-D cultures of human osteoblasts. These were evaluated for protein markers of bone cells. Sequentially alkaline phosphatase, calcium incorporation for matrix mineralisation and then finally osteocalcin expression were detected in cultures. The extracellular matrix was composed of type 1 collagen and as it mineralised, needle shaped crystals were often associated with matrix vesicles initiating mineralisation. In vivo implantation in nude mice showed progression of mineralisation from the inner region outward with peripheral cells in a non-mineralised matrix. Host vessels invaded the implanted cell area. The research has relevance to musculoskeletal tissue engineering.

Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like prolferation, differentiation and death in vitro

There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula in mol% P2O5 (50)-CaO (50-X)-Na2O (X), where X was either 2, 4, 6, 8 or 10 were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion, proliferation and increased cell death in both cell types studied. There was no significant difference in %cell death between the PBGs which was significantly greater than the controls. However, compared to other PBGs, a greater number of cells was found on the 48 mol% CaO which may have been due to either increased adherence, proliferation or both. This composition was capable of supporting osteogenic proliferation and early differentiation and supports the notion that chemical modification of the glass could to lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro

The mammalian nervous system exerts essential control on many physiological processes in the organism and is itself controlled extensively by a variety of genetic regulatory mechanisms. microRNA (miR), an abundant class of small non-coding RNA, are emerging as important post-transcriptional regulators of gene expression in the brain. Increasing evidence indicates that miR regulate both the development and function of the nervous system. Moreover, deficiency in miR function has also been implicated in a number of neurological disorders. Expression profile analysis of miR is necessary to understand their complex role in the regulation of gene expression during the development and differentiation of cells. Here we present a comparative study of miR expression profiles in neuroblastoma, in cortical development, and in neuronal differentiation of embryonic stem (ES) cells. By microarray profiling in combination with real time PCR we show that miR-7 and miR-214 are modulated in neuronal differentiation (as compared to miR-1, -16 and -133a), and control neurite outgrowth in vitro. These findings provide an important step toward further elucidation of miR function and miR-related gene regulatory networks in the mammalian central nervous system. (C) 2010 Elsevier Inc. All rights reserved.

Chromobox protein homolog 3 is essential for stem cell differentiation to smooth muscles in vitro and in embryonic arteriogenesis

Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear.

Establishing a Robust In Vitro Embryonic Stem Cell Differentiation Assay to Monitor the Hematopoietic Potential of DELES Clones

Afin d’effectuer des études fonctionnelles sur le génome de la souris, notre laboratoire a généré une bibliothèque de clones de cellules souches embryonnaires (ESC) présentant des suppressions chromosomiques chevauchantes aléatoires – la bibliothèque DELES. Cette bibliothèque contient des délétions couvrant environ 25% du génome murin. Dans le laboratoire, nous comptons identifier de nouveaux déterminants du destin des cellules hématopoïétiques en utilisant cet outil. Un crible primaire utilisant la benzidine pour démontrer la présence d'hémoglobine dans des corps embryoïdes (EBS) a permis d’identifier plusieurs clones délétés présentant un phénotype hématopoïétique anormal. Comme cet essai ne vérifie que la présence d'hémoglobine, le but de mon projet est d'établir un essai in vitro de différenciation des ESC permettant de mesurer le potentiel hématopoïétique de clones DELES. Mon hypothèse est que l’essai de différenciation hématopoïétique publié par le Dr Keller peut être importé dans notre laboratoire et utilisé pour étudier l'engagement hématopoïétique des clones DELES. À l’aide d’essais de RT-QPCR et de FACS, j’ai pu contrôler la cinétique de différenciation hématopoïétique en suivant l’expression des gènes hématopoïétiques et des marqueurs de surface comme CD41, c-kit, RUNX1, GATA2, CD45, β-globine 1 et TER-119. Cet essai sera utilisé pour valider le potentiel hématopoïétique des clones DELES candidats identifiés dans le crible principal. Mon projet secondaire vise à utiliser la même stratégie rétro-virale a base de Cre-loxP utilisée pour générer la bibliothèque DELES pour générer une bibliothèque de cellules KBM-7 contenant des suppressions chromosomiques chevauchantes. Mon but ici est de tester si la lignée cellulaire leuémique humaine presque haploïde KBM-7 peut être exploitée en utilisant l'approche DELES pour créer cette bibliothèque. La bibliothèque de clones KBM-7 servira à définir les activités moléculaires de drogues anti-leucémiques potentielless que nous avons identifiées dans le laboratoire parce qu’elles inhibent la croissance cellulaire dans plusieurs échantillons de leucémie myéloïde aiguë dérivés de patients. Elle me permettra également d'identifier les voies de signalisation moléculaires qui, lorsque génétiquement perturbées, peuvent conférer une résistance à ces drogues.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Neural differentiation from equine inner cell mass cells in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.