Multiscale technologies for human stem cell culture in suspension: applications for hematopoietic and cardiac differentiation

Stem cell based therapies have been proposed as promising solutions for the regeneration or replacement of diseased tissues, but their potential benefits are limited mainly by scarse cell availability and clinical concern related to cell quality. Aim of the presented research is to develop technologies for human stem cell culture in order to improve the efficiency and the quality of stem cell products. In particular, a bioeractor with a working volume of 10ml/well and a microstructured hydrogel have been designed to perform cultures of human cord blood-derived hematopoietic stem cells (UCB-derived HSCs) in suspension and for controlling cardiac differentiation of human embryonic stem cell-derived embryoid bodies (hES-EBs), respectively. A general aim is to yield the small volumes involved in the proposed technology to span simultaneously a wide range of cell culture conditions which would have prohibitive costs with volumes of hundreds of milliliters. A particularly promising cell-based theraphy is HSC transplantation. UCB-derived CD34+ cells are a source of HSCs potentially very useful for transplantation. However, the current therapeutic use of HSCs from UCB is limited to pediatric patients because of the low cell count from single unit and the difficulty to control the resulting cell phenotype when cells are expanded in vitro. In this scenario, a stirred bioreactor for the expansion of UCB-derived CD34+ cells has been developed in order to investigate the effects of hypoxia on the expression of stem cell markers, as oxygen is recognized to favor stem cell self-renewal in hematopoiesis. The stirring system has been designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 ml/well), is suitable for cell expansion and multi-parametric flow cytometry analyses. The stirred system improves homogeneity within the culture volume thus allowing to control the effective O2 concentration in the cell immediate microenvironment. Flow cytometric analysis show that CD34+ cells cultured in hypoxia conditions are viable for the entire duration of the experiments and maintained a higher expression of c-kit compared to those cultured in standard conditions. In this work, taking advantage of advanced technologies which provided a highly-defined cell culture environment in terms of physical and chemical regulatory signals, we were able to explore the dependence of c-kit expression on oxygen conditions. These results open new perspectives in the study of the mechanisms of interaction between oxygen- and c-kit-dependent pathway at a molecular level. Cell transplantation is emerging as a promising possibility also to replace scarred or nonfunctional myocardium in a diseased heart. Embryonic stem (ES) cells, which self-renew indefinitely while retaining their capacity to differentiate into cell lineages of all three primary germ layers including cardiomyocytes, are one of the most promising source for the development of novel approaches in cell therapy. The use of hES cells is limited by poor progress in the development of robust protocols for cell expansion and differentiation. In this scenario, we developed a culture system for investigating the effects of the local concentration of endogenous factors in the cell microenvironment on hES cell differentiation. In particular, we developed a microwell array on hydrogel as the experimental platform. In our systems, the diffusion of soluble factors through the 3D hydrogel is likely to lead to an accumulation of over times which are relevant for cell proliferation and differentiation. In the described cell culture conditions, aggregations of EBs is avoided and single EBs can be collected for further analyses using a micropipette, without disrupting the culture. In the present study, the differentiation profile of EBs confined in hydrogel microwells of different depths (450 µm and 1 mm) compared to those cultured onto standard ultra low adhesive petri dishes is examined. The analysis of the expression profile of the whole genome by microarray analysis demonstrate that the locally controlled availability of endogenous factors in the microenvironment elicit significant differences in hES differentiation. In particular, confinement of EBs in the microwells promotes the expression of genes involved in the pattern specification process, whereas conventional cultures promote the expression of genes of heart development. Further experiments are required to deeply understand the mechanisms underneath these results and to study the spatial distribution of markers of the three germ layers. The microwell technology may be used to obtain many separated size-controlled EBs and to evaluate the effects of different microenvironments on differentiation. This technology is also suitable to modulate the presentation of morphogenic cues and elucidate the mechanisms of stem cell differentiation. The small amounts of medium required for the obtainment of hundreds of EBs makes this approach a tool to consider when developing in vitro differentiation protocols.L’impiego di cellule staminali per scopi terapeutici è stato proposto come una soluzione promettente per la rigenerazione o la sostituzione di tessuti malati; tuttavia, i potenziali benefici presentano limitazioni legate principalmente alla scarsa disponibilità e ad aspetti di sicurezza clinica connessi alla qualità delle cellule. Scopo del progetto di ricerca è lo sviluppo di tecnologie per la coltura di cellule staminali umane per migliorare l’efficienza e la qualità dei prodotti finali nei processi di espansione e differenziamento. In particolare sono stati progettati un bioreattore a sei pozzetti con un volume di 10ml/pozzetto, per colture in sospensione di cellule staminali ematopoietiche derivate dal cordone ombelicale e un hydrogel microstrutturato per il controllo del differenziamento cardiaco di corpi embriodi derivati da cellule staminali embrionali umane. Un obiettivo generale è sfruttare i piccoli volumi richiesti dalle tecnologie proposte per la valutazione simultanea di un’ampia gamma di condizioni di coltura, soluzione che presenterebbe costi proibitivi con volumi di centinaia di millilitri. Una prospettiva particolarmente promettente per quanto riguarda la terapia cellulare è il trapianto di cellule staminali ematopoietiche. Le cellule CD34+ derivate da sangue di cordone ombelicale costituiscono una risorsa di cellule staminali ematopoietiche utilizzabili per trapianti. Tuttavia, sia per l’esiguo numero di cellule che possono essere ottenute da una singola unità di cordone, sia per la difficoltà di controllare il fenotipo cellulare risultante a seguito di protocolli di espansione in vitro, l’attuale impiego terapeutico di cellule staminali ematopoietiche isolate da sangue di cordone ombelicale è rivolto soltanto a pazienti pediatrici. In questo contesto, è stato realizzato un bioreattore con un sistema di agitazione per espandere cellule CD34+ derivate da sangue di cordone ombelicale e studiare gli effetti dell’ipossia nell’espressione di marcatori di staminalità. E' noto infatti che l’ossigeno favorisce il rinnovamento cellulare durante l’ematopoiesi. Il sistema di mescolamento è stato realizzato in modo da poter essere applicato su di una piastra convenzionale a sei pozzetti, sia per uniformità con i sistemi di coltura statici convenzionali che per la conservazione dei protocolli di coltura. I volumi del bioreattore (10 ml/pozzetto) sono adatti per l’espansione delle cellule e per analisi multiparametriche mediante tecniche di citofluorimetria. Il sistema di mescolamento favorisce l’omogeneità all'interno del volume di coltura, permettendo così di poter controllare la concentrazione di ossigeno disciolto nell’immediato microambiente cellulare. Analisi di citofluorimetria evidenziano che cellule CD34+ coltivate in condizioni di ipossia rimangono vitali nel corso dell’intera durata degli esperimenti e presentano una maggiore espressione del c-kit rispetto alle cellule coltivate nelle condizioni di coltura convenzionali. In questo studio, sfruttando il vantaggi offerti dall’impiego di tecnologie avanzate che hanno consentito di ottenere un ambiente di coltura altamente definito da un punto di vista chimico – fisico, siamo stati in grado di esplorare la relazione tra l’espressione di c-kit e le condizioni di ossigeno. Questi risultati aprono nuove prospettive nello studio dei meccanismi di interazione tra l’ossigeno e le vie biologiche dipendenti da c-kit a livello molecolare. Il trapianto di cellule sta emergendo come possibilità promettente anche per la sostituzione di tessuto cardiaco danneggiato o malfunzionante in un cuore malato. Le cellule staminali embrionali umane, in grado di rinnovarsi indefinitamente mantenendo inalterata la loro capacità di differenziare in tutti i tipi cellulari dei tre foglietti germinativi, compresi i cardiomiociti, rappresentano una delle fonti più promettenti per lo sviluppo di nuovi approcci alla terapia cellulare. Tuttavia, L’impiego di cellule staminali embrionali umane è limitato dal lento progresso nello sviluppo di protocolli per l’espansione e il differenziamento in vitro. In questo contesto, abbiamo progettato un sistema di coltura che ci consentisse di esplorare gli effetti della concentrazione locale di fattori endogeni nel microambiente cellulare sul differenziamento delle cellule staminali embrionali umane. In particolare, abbiamo sviluppato un array di micropozzetti su hydrogel come piattaforma sperimentale. Nel nostro sistema, è possibile ipotizzare che la diffusione di fattori solubili nell’hydrogel tridimensionale porti a un accumulo dei fattori stessi in tempi rilevanti per i processi di proliferazione e differenziamento cellulare. Nelle condizioni di coltura descritte, l’aggregazione di corpi embriodi è impedita e singoli corpi embrioidi possono essere isolati e raccolti con una micropipetta per ulteriori analisi, senza interferire con la coltura. Nello studio, è stato analizzato il profilo di differenziamento dei corpi embrioidi confinati nei microwells di diversa profondità (450 μm e 1 mm) a confronto con quello delle cellule coltivate su piastre non aderenti convenzionali. L’analisi dell’espressione genica dell’intero genoma mediante microarray dimostra che il controllo della disponibilità locale di fattori endogeni nel microambiente cellulare induce differenze significative nel differenziamento delle cellule staminali embrionali umane. In particolare, il confinamento dei corpi embrioidi nei micropozzetti promuove l’espressione di geni coinvolti nei processi di specificazione della compartimentazione dell’embrione, mentre sistemi di coltura convenzionali promuovono l’espressione di geni coinvolti nello sviluppo del cuore. Ulteriori esperimenti sarano necessari per comprendere in modo approfondito i meccanismi alla base dei risultati ottenuti e per studiare la distribuzione spaziale di marcatori dei tre foglietti germinativi. La tecnologia dei micropozzetti può essere utilizzata per ottenere un alto numero di corpi embrioidi separati e di dimensione definita e per valutare gli effetti del diverso microambiente sul differenziamento delle cellule. Questa tecnologia è inoltre adatta per modulare la disponibilità di fattori morfogenici e chiarire i meccanismi del differenziamento delle cellule staminali. I piccoli volumi richiesti per ottenere centinaia di corpi embrioidi rendono questo approccio uno strumento da prendere in considerazione quando si vogliano sviluppare protocolli di differenziamento in vitro

Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2)O(2). Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2)O(2), but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development

Design of a stirred multiwell bioreactor for expansion of CD34(+) umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O(2) concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O(2) solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O(2) concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O(2) for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O(2) , using 21%-O(2) cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level

Controlled cardiac differentiation of human embryonic stem cell-derived embryoid bodies in scalable bioreactors

none7noneZAGALLO M; LUNI C; SERENA E; CIMETTA E; ZATTI S.; GIOBBE G; ELVASSORE NZagallo, Monica; Luni, Camilla; Serena, Elena; Cimetta, Elisa; Zatti, S.; Giobbe, GIOVANNI GIUSEPPE; Elvassore, Nicol

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification

Characterization of microstructured hCMs culture.

<p><b>A</b> - Cardiac markers immunofluorescence; cTnT, connexin 43, α-actinin, NKX2.5. Nuclei were counterstained with hoechst. <b>B</b> - Immunofluorescence against and adult isoform of cardiac Troponin I of T39 hCMs and adult/fetal isoform of cardiac Troponin T of T39 hCMs, nuclei were counterstained with hoechst. Scale bars: A: 75 µm and 25 µm for NKX2.5; B: 10 µm.</p