Human Embryonic-Derived Mesenchymal Progenitor Cells (hES-MP Cells) are Fully Supported in Culture with Human Platelet Lysates

Publisher's version (útgefin grein)Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL). View Full-TextThis research was partially supported by the IRF Fund (grant number 152388-052). The authors want to thank the Landspitali University Hospital Research Fund for partially funding the project. We would also like to thank Ragna Landro and Bjorn Hardarsson for technical support and Kristine Wichuk and Steinunn Thorlacius for editing and reviewing the manuscript. We are grateful to the Blood Bank, Reykjavik, Iceland and Takara Bio Europe AB (previously Cellartis AB), Gothenburg, Sweden for providing the platelets and hES-MP cells, respectively, used in the study.Peer Reviewe

Effects of amotosalen treatment on human platelet lysate bioactivity: A proof-of-concept study.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadBackground: Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation. Methodology/principal findings: Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates. Conclusion/significance: These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing

Notkun útrunnina blóðflaga til fjölgunar á mesenkímal stofnfrumum

Mesenchymal stromal cells (MSC) are promising candidates for cellular therapy due to their ability to regenerate bone and cartilage and modulate immune responses. The use of MSC in cellular therapy is problematic due to the need for fetal bovine serum (FBS) during ex vivo expansion of the cells. The use of FBS may lead to detrimental side effects for patients such as cross-species viral infections and severe immune responses. Alternative cell culture supplements are therefore needed. Lysates derived from platelets have been suggested, mainly due to the abundance of various growth factors and cytokines that are found in platelet granules. Platelets to make lysates can be obtained from blood banks. Blood banks, however, do not possess a large surplus of platelets since they face a shortage of platelet donors. Still, a significant number of platelet concentrates expire annually in blood banks and are discarded. These expired platelets could be used as an alternative to FBS to make lysates in an economical and effective way. Furthermore, the resources of blood banks would be utilized in a more efficient manner. Human embryonic-derived mesenchymal progenitor (hES-MP) cells have been envisioned as a future source of MSC which, for the same reason as MSC, also require serum-free culture supplements. In this thesis, we evaluated the suitability of expired platelets and expired pathogen-inactivated platelets to support the growth, phenotype, immune function, and tissue formation of MSC and hES-MP cells. Platelet lysates from expired platelets supported MSC and hES-MP growth, phenotype, and tissue formation to an equal or greater extent than lysates from fresh platelets or FBS. Platelet lysates supported osteogenic differentiation particularly well, causing upregulation of alkaline phosphatase activity, tissue mineralization, and osteogenic gene expression. Similar observations were obtained for platelet lysates from expired pathogen-inactivated platelets which supported MSC in all aspects evaluated. Furthermore, adding platelet lysates from expired pathogen-inactivated platelets to the tissue differentiation media enhanced both osteogenic and chondrogenic differentiation of MSC and hES-MP compared to FBS. In both platelet lysate and FBS, hES-MP cells proliferated faster than MSC and reached higher cell numbers in shorter time. However, hES-MP failed to suppress immune cell proliferation, while MSC did so effectively. Both cell types, nevertheless, differentiated successfully toward osteogenic, chondrogenic, and adipogenic lineages independent of the culture supplement used. We conclude that lysates from expired platelets, pathogen-inactivated or not, successfully support the expansion of MSC and hES-MP while also allowing the cells to maintain their capacity to modulate immune responses and differentiate. Expired platelets therefore represent a feasible and attractive source material to make platelet lysates for FBS replacement.Mesenkímal stofnfrumur úr beinmerg (MSC) lofa góðu fyrir notkun í vefjalækningum sökum hæfni þeirra til að mynda vefi stoðkerfisins og til að stýra ónæmissvari. Notkun þeirra eru þó vandasöm vegna þess að kálfasermi þarf til að rækta þær utan líkamans. Kálfasermi er óæskilegt því það getur haft skaðleg áhrif í för með sér, svo sem hættu á dýrbornusmiti og ónæmis- og bólgusvari í frumuþega. Nauðsynlegt er að finna aðra lausn sem er ekki upprunnin úr dýrum, leysir kálfasermi af hólmi og styður við MSC frumur í rækt. Blóðflögulausnir úr blóðflögum manna hafa verið ræddar í þessu samhengi sökum þess hve ríkar þær eru af vaxtarþáttum og frumuboðum sem finnast í seytiögnum þeirra. Blóðflögur sem má breyta í blóðflögulausnir fást hjá blóðbönkum. Blóðbankar eiga hinsvegar ekki umframmagn af blóðflögum til að láta af hendi þar sem þeir búa nú þegar við skort á blóðgjöfum. Engu að síður þarf að farga töluverðu magni af blóðflögum árlega vegna þess að þær renna út. Útrunnar blóðflögur væri hægt að nota til að útbúa ræktunarlausnir á hagkvæman, ódýran og skilvirkan hátt sem staðgengil kálfasermis fyrir frumuræktir. MSC frumur sérhæfðar frá stofnfrumum úr fósturvísum (hES-MP) hafa líka verið skoðaðar sem möguleiki fyrir vefjalækningar og því einnig mikilvægt að finna ræktunarlausnir fyrir þær sem eru ekki upprunar frá dýrum. Í þessari rannsókn skoðuðum við hæfni útrunnina blóðflagna og útrunnina smithreinsaðra blóðflagna frá Blóðbankanum til að styðja við MSC og hES-MP frumur í rækt. Frumuvöxtur, tjáning á yfirborðssameindum, þátttaka í ónæmissvari og hæfni þeirra til að mynda vefi stoðkerfisins var skoðuð sérstaklega. Blóðflögulausnir úr útrunnum blóðflögum voru jafngildar eða betri en kálfasermi og blóðflögulausnir úr ferskum blóðflögum þegar frumuvöxtur, tjáning á yfirborðssameindum og vefjamyndun var skoðuð hjá MSC og hES-MP frumum. Blóðflögulausnir hentuðu sérstaklega vel til að styðja við beinmyndun sem sást með aukinni virkni alkalísks fosfatasa, útfellingu steinefna í vef og aukinni genatjáningu fyrir beinmyndun. Sambærilegar niðurstöður komu fram við notkun á blóðflögulausnum úr útrunnum smithreinsuðum blóðflögum. Þegar slíkum blóðflögulausnum var bætt út í æti fyrir vefjasérhæfingu kom fram aukin bein- og brjóskmyndun umfram það sem sást við notkun á kálfasermi. hES-MP frumur, bæði í blóðflögulausnum og kálfasermi, fjölguðu sér hraðar heldur en MSC frumur. hES-MP frumur gátu hinsvegar ekki dregið úr fjölgun ónæmisfruma líkt og MSC frumur gera. Báðar frumutegundir mynduðu þó bein, brjósk og fituvef. Blóðflögulausnir úr útrunnum blóðflögum, smithreinsuðum eður ei, henta sem ræktunarlausnir fyrir MSC og hES-MP frumur án þess að draga úr hæfni þeirra til frumufjölgunar, þátttöku í ónæmissvari eða vefjamyndun. Útrunnar blóðflögur eru því ákjósanlegur efniviður fyrir blóðflögulausnir sem nota má í stað kálfasermis.This project was funded by The Icelandic Research Council (RANNÍS) and Landspítali – University Hospital of Iceland research fund (Vísindasjóður)

Paul Petroff as the Prince and Tamara Toumanova as the Queen of the Swans, in Le lac des cygnes, the Original Ballet Russe, Australian tour, His Majesty's Theatre, Melbourne, 1940 [picture] /

From: Le lac des cygnes (Swan lake) : choreographic poem in one act / music by Peter Ilich Tchaikovsky; Inscription: "3P/3".; Part of the collection: Hugh P. Hall collection of photographs, 1938-1940.; Performed March and April 1940.; Condition: Poor, silvering.; Choreography after M. Petipa ; scenery and costumes by C. Korovine ; scenery executed by O. Allegri.; Also available in an electronic version via the internet at: http://nla.gov.au/nla.pic-vn4175660. One of a collection of photographs taken by Hugh P. Hall of 28 ballet productions performed by the Covent Garden Russian Ballet (toured Australia 1938-1939) and the Original Ballet Russe (toured Australia 1939-1940). These are the second and third of the three Ballets Russes companies which toured Australasia between 1936 and 1940. The photographs were taken from the auditorium during a live performance in His Majesty's Theatre, Melbourne and mounted on cardboard for display purposes. For conservation and storage, the photographs have been demounted. The original arrangement of the photographs has been recorded, and details are available from the Pictures Branch of the National Library

Mesenchymal stem cell morphology during expansion and differentiation.

<p>MSC were stained with crystal violet after expansion in media containing 10% of FBS, HPLF or HPLO for three passages (A–C). MSC cultured in HPLF or HPLO developed more spindle shaped morphology than cells cultured in FBS, proliferated faster and left circular areas containing no cells between them. Differentiation towards osteoblasts, adipocytes and chondrocytes was initiated after expansion in the three supplements. After 28 days of osteogenic differentiation mineralization in the culture could be visualized with a Von Kossa staining (D–F). Successful adipogenic differentiation was confirmed with Oil red O staining (G–I) and after 28 days of chondrogenic pellet cultures, the pellets were sectioned and stained with toludine blue staining (J–L). Pictures representative of three experiments.</p

Cumulative population doublings of MSC after culture in FBS, HPLF or HPLO.

<p>MSC were cultured in culture media supplemented with 10% of FBS, platelet lysate from fresh platelet concentrates (HPLF) or lysate from expired platelet concentrates (HPLO). Population doubling assay was performed at the end of every passage for a total of six passages (P1–P6, n = 3). MSC cultured in either HPLF or HPLO consistently had higher numbers of population doublings at the end of every passage compared to MSC cultured in FBS. By the end of the sixth passage cumulative population doublings were 13.77±0.77 CPD for HPLO, 12.77±0.12 CPD for HPLF and 9.50±1.14 CPD for FBS supplemented cultures, respectively.</p

Expression of osteogenic marker genes during osteogenic differentiation of MSC.

<p>Osteogenic differentiation was initiated after MSC expansion in 10% of FBS, HPLF or HPLO. Expression of osteogenic marker genes was evaluated after 7 and 21 days of osteogenic differentiation and is represented with graphs for each gene (A–C, n = 3) and a heat-map (D) where green shades represent lower levels of expression and red shades higher levels of expression. <i>ALP</i> was evenly expressed throughout the differentiation (A,D) while the expression of <i>SPP1</i> increased from day 7 to day 21 (B,D), irrespective of the culture supplements the MSC had previously been exposed to (p≤0.01). Expression of <i>RUNX2</i> decreased from day 7 to day 21 for all cultures (C–D), still expression of <i>RUNX2</i> in cultures originating from HPLO treated MSC was higher at both time-points compared to FBS and also at day 21 for HPLF compared to FBS (p≤0.05). * = p≤0.05, ** = p≤0.01.</p

Reduction of mononuclear cell proliferation during co-culture with MSC.

<p>Mononuclear cells (MNC) were stimulated with phytohemaglutinin (PHA) and then cultured with or without MSC that had been previously expanded in media containing 10% of FBS, HPLF or HPLO. MNC proliferation was significantly less when co-cultured with MSC than if cultured on their own (p≤0.05, n = 3). The reduced proliferation was seen irrespective of the culture conditions the MSC had previously been exposed to. * = p≤0.05.</p

Increased enzymatic activity of alkaline phosphatase during osteogenic differentiation.

<p>MSC that had previously been cultured in media containing 10% of FBS, HPLF or HPLO underwent osteogenic differentiation. ALP activity was evaluated after 7 and 14 days of differentiation. ALP activity increased significantly from day 7 to day 14 independent of the type of culture conditions the MSC had been exposed to prior to osteogenic differentiation (p≤0.05, n = 3). * = p≤0.05.</p

Surface antigen expression of MSC cultured with 10% FBS, 10% HPLF or 10% HPLO.

<p>Surface antigen expression of MSC cultured with 10% FBS, 10% HPLF or 10% HPLO.</p