large-scale production of murine embryonic stem cell-derived osteoblasts

7/28/2019 Large-Scale Production of Murine Embryonic Stem Cell-Derived Osteoblasts

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passaging and harvesting protocols are time consuming and labor-intensive. Static tissue culture systems also lack continuous moni-toring and tight control of the culture microenvironment, whichcould lead to spontaneous stem cell differentiation and/or cultureheterogeneity, and typically employ the use of animal-derivedproducts [13e21]. Furthermore, the production of mineralizedextracellular matrix from ESC-derived osteogenic cells createstremendous difficulties when attempting to harvest the cells fromadherent static cultures, and leads to extensive cell loss.

Exposure of ESCs to serum-containing media poses a majorconcern for downstream clinical applications because of the effectson ESC immunogenicity and potential pathogenicity. Serum issubject to considerable lot-to-lot variability and may containunidentified pathogens, prions and viruses [22]. Moreover, forclinical applications, it will be important to avoid immune-basedrejection responses including xenogeneic responses induced bythe presence of animal-derived components [23].

Finally, a three dimensional (3D) culture system more closelyresembles the physiological environment and has been shown topromote lineage-specific differentiation of several types of cells[24]. In considering the high demand for alternative treatments formusculoskeletal diseases and the limited supply of cells, as well as

concerns surrounding differentiation efficiencies, it is clear that thedevelopment of a robust, reproducible bioprocesses for theproduction of clinically relevant quantities of osteoblasts andchondrocytes is necessary.

First introduced by van Wezel in 1967 to mass produce viralvaccines and biological cell products using mammalian cells [25],microcarriers have been successfully used for the large-scaleproduction of a variety of biological products [26]. Microcarriersprovide immense surface areas for attachment and expansion ofanchorage-dependent cells in stirred tank reactors. The use ofmicrocarriers offers a number of advantages including lower totalcosts, ease of harvesting and downstream processing procedures,ease of scale-up, and an overall reduction in the space required fora given-sized operation due to the high growth surface area per

unit volume of reactor [27].Microcarriers have typically been used in the production of viral

vaccines using VERO cells, and the production of recombinanttherapeutics using Chinese Hamster Ovary (CHO) and BabyHamster Kidney (BHK) cells [28e30]. In additionto their capacitytoserve as scaffolds for the propagation of anchorage-dependent andanchorage-preferred cells, microcarriers are also efficacious toemploy as delivery vehicles for implantation of the cells in vivo.They have been used for the culture of pancreatic islet cells [31]mesenchymal stem cells [32e34] and fibroblast cells [35e37]amongst others.

Despite the potential benefits of producing clinically relevantquantities of osteoblasts and chondrocytes for cellular therapies, todate however, there have been no studies on the productionof ESC-

derived osteoblasts or chondrocytes in a feeder-free, serum-freesystem using microcarrier scaffolds. To this end, we set out todevelop an effective system for the large-scale production of ESC-derived cells on microcarriers under feeder-free and serum-freeconditions in stirred suspension bioreactors.

2. Materials and methods

2.1. Mouse embryonic stem cell line

Unless otherwise stated, all cell-handling procedures were conducted ina sterile laminar flow hood using aseptic techniques and all ESC cultures werecarried out in a humidified, 37 C and 5% CO2 incubator. Murine D3 ESCs (ATCC,American Type Culture Collection, Rockville, MD, USA) were used in all cultures. Allbioreactor cultures were performed in duplicate and all cell counts were performed

on duplicate samples.

2.2. Culture medium

2.2.1. Maintenance medium

Murine ESC maintenance medium consisted of Dulbeccos Modified EagleMedium with high glucose (DMEM, Invitrogen, Carlsbad, California USA. Cat#10569-010) supplemented with 15% Knockout-Serum Replacement (KSR, Invi-trogen, Cat# 10828-018), 1% v/v non-essential amino acids (NEAA, Invitrogen,Carlsbad, California USA Cat# 11140-050), 0.2% (v/v) b-mercaptoethanol (Invitrogen,Carlsbad, California USA Cat# 21985-023) and 1% v/v penicillin/streptomycin (Pen/

Strep, Invitrogen, Cat# 15140-122).

2.2.2. Osteogenic medium

Osteogenic medium (OM) consistedof maintenancemedium supplemented with105 M dexamethasone (SigmaeAldrich, Ontario Canada. Cat# D4902), 50 mg/mLascorbic acid (SigmaeAldrich, Ontario Canada. Cat# A4403), and 5 mM b-glycer-ophosphate (SigmaeAldrich, Ontario Canada. Cat# G9891).

2.2.3. Chondrogenic medium

There were two types of chondrogenic media which were used in separatecultures; (i) chondrogenic medium 1 (CM1) consisted of DMEM (Invitrogen, Carls-bad, California USA. Cat# 12100-061), supplemented with 15% Knockout-SerumReplacement (KSR, Invitrogen, Cat# 10828-018), 1% non-essential amino acids(NEAA, Invitrogen, Carlsbad, California USA. Cat# 11140-050), 1% penicillin/strep-tomycin (Pen/Strep, Invitrogen, Cat# 15140-122), 1 mM Sodium Pyruvate (100,Gibco, Carlsbad, California USA. #11140-050), 100 mM b-mercaptoethanol (550,Gibco #21985-0230), 50 mg/mL Ascorbic acid (SigmaeAldrich, Ontario Canada),10 ng/mL BMP-2 (BMP-2, PeproTech # 120-02), 10 ng/mL TGFb1 (TGFb1, PeproTechRocky Hill, NJ, USA. Cat# 100-21C) and 1% of an insulinetransferrineseleniumcomplex (ITS, Invitrogen, Ontario Canada #51500-056), and (ii) chondrogenicmedium 2 (CM2) consisted of all of the components in CM1 minus the TGFb1.

2.3. Mouse embryonic stem cell cultivation

2.3.1. Microcarrier preparation and cell expansion

For microcarrier preparation, 0.1 g CultiSpher S (Percell Biolytica, storp,Sweden), a highly cross-linked type A porcine-derived gelatin macroporousmicrocarrier was weighed dry and initially hydrated separately in 50 mL of Ca2,Mg2 e free phosphate buffer saline (PBS, Invitrogen) overnight at room tempera-ture. The supernatant was then removed and the microcarriers were washed twicein fresh PBS and sterilized by autoclaving at 120 C for 30 min. After sterilization,microcarriers were equilibrated in 20 mL of culture medium with LIF in NDS 125 mLbioreactors (NDS Technologies, Palo Alto, California USA, Cat# 264501-125) at 37 Cand 5% CO2. After 1 h, medium volume was increased to 50 mL and cultivation inbioreactors was performed at 60 rpm, 5% CO2 and37 C. ESCs previously thawedand

expanded inT-75 tissue culture vessels were used to seed sterilized microcarriers ata 5:1 cell to bead ratio. Cultures were then intermittentlystirred (3 minof stirring at60 rpm followed by 27 min off) during the first 8 h of culture. The medium wastopped-upto a finalvolume of 100 mL andstirred continuouslyat 60 rpm for 6 days.Medium was changed every second day during the expansion phase.

2.3.2. Differentiation to osteoblasts and chondrocytes

Osteogenic cultures consisted of maintenancemedium replaced with osteogenicmedium; (i) a single cell inoculum without microcarriers (SC-MCs), agitated at100 rpm, (ii) single cell inoculum plus empty sterilized microcarriers (SC MCs)agitated at 60 rpm, (iii) cell-loaded inoculum microcarriers from day 6 expansion,induced into differentiation without dissociation (CL MCs) agitated at 60 and100 rpm. For all conditions, differentiation was carried out for 30 days.

For chondrogenic differentiation, maintenance medium was replaced with CM1and bioreactors were seeded with day 6 cell-loaded microcarriers (CL MCs) andagitated at 60 or 100 rpm. On day 5, CM1 was replaced with CM2 for the remainderof the differentiation. For all conditions, differentiation proceeded for a total of

30 days.

2.4. Cell harvest

To harvest cells from microcarrier cultures, 2.0 mL duplicate samples wereretrieved from spinners and placed in 15 mL centrifuge tubes. The supernatant wasremoved and the microcarriers rinsed twice with PBS and trypsinized with 0.25%Trypsin-EDTA (Invitrogen, Cat# 25200-056) for 15 min, with mechanical dissocia-tion every 5 min. CultiSpher S microcarriers completely degraded upon trypsini-zation. The resulting cell suspension was preserved for later analyses.

2.5. Immunocytochemistry and flow cytometry

For immunocytochemistry, cells were washed twice with PBS and fixed with 4%Paraformaldehyde (Sigma) in PBS (pH 7.4) for 15 min at room temperature. Afterwashing 3withPBS, cells werepermeabilized with0.5% Saponin (Sigma, G-7900) in1PBS/1%BSAfor15minatroomtemperatureandthenblockedwith3%bovineserum

albumin(Sigmae

Aldrich,Ontario,Canada. Cat.# A9418) in 1

PBSfor30min.Thecells

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were then incubated overnight at 4 C with primary antibodies against osterix,osteocalcinand collagenI forosteoblasts,andaggrecan andcollagenII forchondrocytes(allfromSantaCruzBiotechnology,SantaCruz,CA).Thenextday,thecellswerewashed3with PBS for 5 min and incubated in the dark with secondary antibodies for 1 h atroomtemperature.Secondaryantibodiesused wereFITC conjugated donkey anti-goatantibodies (Santa Cruz Biotechnology, Santa Cruz, CA or Jackson ImmunoResearchLaboratories). Flowcytometry analysiswas carriedout on harvesteddifferentiatedcellsusing a FACS Calibur instrument and CellQuest software from Becton Dickinson. Eachsample was run twice, with 10,000 events registered per run. The data was gated

between a forwardscatter(FSC-H)of 200 and800, as to excludecelldebrisand clumpsof cells respectively. Controls were stained with secondary antibodies only.Immunoassaying of intact colonized microcarrierswas performed and visualized

using a Carl Zeiss laser scanning confocal microscope. For immunoassaying, intactcolonized microcarriers were collected from spinners and let to settle for a fewminutes in a 15mL centrifuge tube. Thesupernatantwas collectedand disposed.Thebeads were then preserved in 4% PFA overnight. The next day, microcarriers werewashed twice in 1 PBS and permeabilized with a 0.5% saponin in 1 PBS overnightat 4 C. Samples werelater stained overnight withprimary antibodiesagainst osterix,osteocalcin and collagen I for osteoblasts, and Aggrecan and Collagen II for chon-drocytes (all from Santa Cruz Biotechnology, Santa Cruz, CA). A FITC conjugatedsecondaryantibody was used thenextday to stainthe cells.Afterstaining, theloadedmicrocarriers were then mounted on slide with spacers using a mountant (9:1,glycerol: PBS) and the sides were sealed with clear nail polish and later visualized.

2.6. Histological analyses of differentiated colonized microcarriers

At the end of the differentiation, day 29 cell-loaded CultiSpher S microcarrierswere processed on the Tissue-Tek VIP 6 (Torrance, California USA) automated

processor for various histological analyses. Initially, samples were embedded intomolds, creatingparaffin blocks. The blocks were then cut on a Leica 2125 microtome(Leica Microsystems, Ontario Canada) at 4 mm and placed on slides for staining andanalyses. Stained slides were visualized under a Leica DMIL inverted microscope.

Differentiated samples were assessed for calcium and mineral deposition byalizarin red and von Kossa staining for osteogenic differentiation, and alcian blue forcartilage. All conditions were tested to determine whether mineralization wasenhanced or impeded by agitation or inoculum form. Samples tested included; (i)cell-loaded osteogenic induced microcarriers agitated at 100 rpm, (ii) cell-loaded

osteogenic induced microcarriers, agitated at 60 rpm, (iii) single cell osteogenicinduced microcarriers, agitated at 60 rpm and (iv) cell-loaded chondrogenicinducedmicrocarriers, agitated at 60 or 100 rpm. Positive controls used for histologicalanalyses were bovine bone tissue samples. Chondrogenic-differentiated cell-loadedCultiSpher S microcarrier samples were used as negative controls for mineraldeposition, while the osteogenic-differentiated cell-loaded CultiSpher S micro-carrier samples were used as negative controls for ECM deposition and cartilageformation.

2.6.1. von Kossa

von Kossa staining was used to visualize mineralization. The von Kossa staindoes not react with calcium but rather reacts with phosphate in the presence ofacidic materials [38]. Mineralization was determined by black or brown depositionsfrom the reaction between silver ions with phosphate ions.

2.6.2. Massons trichrome

Massons Trichrome stain was used to examine the types of tissue presentwithin the excised tissue. Red colored tissuewas identified as skeletal muscle tissue,blue or green coloration was identified as collagen and bone.

Fig.1. Process schematic shows culture, differentiation and transplantation of embryonic stem cell-derived osteoblasts and chondrocytes into animal models. Upon isolation, ESCs

were scaled-up in suspension bioreactors in serum-free medium and then differentiated to osteoblasts or chondrocytes in suspension bioreactors with or without the use of

microcarriers to obtain relevant numbers of cells. Differentiated cells were then harvested and transplanted as full osteogenic aggregates without microcarriers (from SC-MCs) or as

cell-loaded microcarrier scaffolds (CL

MCs).

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2.6.3. Alcian blue staining

Alcian blue dye was used to identify the presence of glycosaminoglycans (GAG)in the chondrogenic cell-loaded microcarriers (CL MCs). These samples wereprepared for histology as described above. Cartilage nodule formation was evaluatedon prepared slides from day 29 of chondrogenic culture.

2.6.4. Alizarin red S staining

To prepare cells for Alizarin Red staining, differentiation media was rinsed offeach well with 2.0 mL PBS. After rinsing, 2.0 mL of buffered formalin was added to

each well and incubated for 1 h at room temperature. The formalin was then dis-carded and the wells were rinsed twice with de-ionized (DI) water. After rinsing,2.0 mLof 1%AlizarinRedS solution was added tothe wells and incubatedfor20 minat roomtemperatureand the wells were rinsed twice with 2.0 mL of DI water. Slideswere captured using a Zeiss inverted microscope.

2.7. In vivo implantation of esc-derived cells

To assess the tumorigenicity of the differentiated cells in vivo, day 15 osteogenicaggregates (obtained from the SC-MC inoculum) as well as day 15 osteogenic cell-loaded microcarriers (CL MCs) were separately injected subcutaneously intosevere combined immuno-deficient (SCID) mice (Taconic, Albany, New York. USA),using a 26 gauge syringe. Cells were not removed from the aggregates or micro-carriers. Mice were housed in the single-barrier animal facility at the University ofCalgary. The animals were euthanized after 6 weeks, and a second set of mice wereeuthanized after 8 weeks post-implantation. The tissue at the point of implantationwas dissected and fixed in 10% formalin for histological analysis. After dehydrationin ascending concentrations of ethanol and xylene, the specimens were embeddedin paraffin, and sections were then deparaffinized, hydrated, and stained for histo-logical analyses.

Tumorigenicity of the cells was also assessed by implanting osteogenic cell-loaded microcarriers into burr-hole fractures created in the tibiae of osteoporotic(OVX)mice.One burr-hole fracture wasinduced in theleft proximal tibiaein 5 Sv129strain mice (strain-matched to the ESCs), as described by Taiani et al. (2010). Eachmouse was first ovariectomized, then the fracture was induced and then cell-loadedmicrocarriers were immediately implanted into the fracture. For implantation,approximately twenty day 25 cell-loaded CultiSpher S microcarriers were liftedfroma Petri dish usinga scalpel blade,transferred to the injury site and then pusheddown into the burr-hole with a 27-gauge needle. Bone histomorphometry wasassessed at day 0 and at 1 week, 2 weeks and 4 weeks following implantation usingin vivo micro-CT imaging at an isotropic resolution of 15 mm (Micro-CT 35, ScancoMedical, Brttisellen, Switzerland). All surgical and animal care procedures wereapproved by the University of Calgary Animal Care Committee.

2.7.1. Statistical analysis

Statistical analyses of the growth kinetics and FACS analyses data were con-ducted using the one-way ANOVA (analysis of variance). Results were consideredstatistically significant ifp < 0.05.

3. Results

3.1. Effect of inoculum form and agitation rate on differentiation

ESCs were expandedfirst in static tissue cultureflasks for 3 days(Day9 to6) and then in bioreactors in serum fee medium duringthe next 6 days (Day 6 to Day 0) as shown in Fig. 1. Fig. 2 (aed)shows the morphology of cells during the expansion period onCultiSpher S microcarriers, and the growth kinetics are representedin Fig. 2 (e). Following expansion, cultures were induced intoosteogenic or chondrogenic differentiation (Day 0), while the cellswere in the exponential growth phase. The effects of inoculumformand agitation on differentiation in serum-free medium wereinvestigated.

To investigate the effect of inoculum form, one set of bioreactorswere seeded with cell-loaded microcarriers and agitated at 60 rpm(CL MCs). Here, maintenance medium was completely replacedwith osteogenic medium. Another set of bioreactors was filled withempty sterilized microcarriers, equilibrated with osteogenicdifferentiation media, and then seeded with a single cell inoculum(SC MCs). Here again, cultures were agitated at 60 rpm.

The effect of agitation rate was also investigated for osteogenic

and chondrogenic-differentiated cultures. Here, bioreactors withosteogenic or chondrogenic differentiation media were inoculatedwith cell-loaded microcarriers and cultures were agitated either at60 rpm (0.35 Pa maximum shear stress) or 100 rpm (0.60 Pamaximum shear stress). Samples were collected at various timeintervals to assess the effect of agitation on differentiation.

3.2. Culture morphology

The appearance of the cultures is shown over the course of the30 days in Fig. 3 (aeo). Upon osteogenic induction, differentiatedcells began to mineralize, as seen in Fig. 3 (aec, feh, kem). By day10 of induction, more agglomeration could be observed in thesingle cell inoculum (Fig. 3 f), compared to the cell-loaded inoc-

ulum (Fig. 3 g). This is not unusual as ESCs have been known toaggregate when cultured in suspension [39e42]. The presence ofmore cells on the surface of the microcarriers at the start ofdifferentiation, as indicated by the black arrows in Fig. 3a may have

Fig. 2. Photomicrographs represent day 0 and day 4 progression of expansion for murine ESCs cultured on CultiSpher S microcarriers. Top row (a, b) represents day 0 brightfield and

DAPI images, respectively, while bottom row (c, d) represents day 4 brightfield and DAPI images, respectively. A representative growth curve for murine embryonic stem cells,

cultured on CultiSpher S microcarriers in serum-free media is shown in (e). For all conditions, bioreactors were inoculated with single cells at 60,000 cells/mL, agitated at 60 rpm

and incubated at 37

C and 5% CO2. Scale bars represent 125 mm.



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contributed to agglomeration within the cultures. There was aneffect of agitation rate that became more apparent by day 30.Specifically, the cultures at 100 rpm had smaller cell clusters anda narrower size distribution than the same cultures at 60 rpm.

Chondrogenic induction proceeded with cell-loaded micro-

carriers (CLMCs) agitated at 60 rpm and at 100 rpm. By day 30 ofcultivation, cellular outgrowths could be observed in the cultures(Fig. 3 n, o). However, the size of the outgrowths in the 60 rpmcultures were larger than those in cultures agitated at 100 rpm,suggesting that higher agitation rates led to better size control.

3.3. Flow cytometry and immunocytochemistry of harvested cells

from CultiSpher S microcarrier cultures

Murine embryonic stem cells induced into osteogenic differen-tiation on CultiSpher S macroporous microcarriers were completelydissociated into single cells after treatment with trypsin, whichdissolved the microcarriers. Flow cytometry analyses of osteogeniccells harvested at various time points of differentiation showed

high expression (over 70%) of osterix, osteocalcin and collagen type

I, as shown in Fig. 3 (per). Osterix, an early transcription factor inosteogenic differentiation, was expressed on day 5 of differentia-tion and peaked by day 14 for all conditions tested. Collagen type Iand osteocalcin, the major noncollagenous component of bonematrix were expressed by day 14 of differentiation. Markers of

osteogenic differentiation, including osterix, collagen type I andosteocalcin, for both forms of inoculation (SC MCs or CL MCs),showed no statistically relevant difference in the fraction of cellsexpressing the osteogenic markers as seen in Fig. 3 (p, q & r).Analyses of osteogenic or chondrogenic cultures agitated at100 rpm versus 60 rpm, showed no statistically relevant differencein the number of cells expressing the markers analyzed, as seen inFig. 3 (p, q & r) for osteoblasts or Fig. 3 (s & t) for chondrocytes.

Whole intact osteogenic and chondrogenic cell-loaded micro-carriers were probed for markers of osteogenic and chondrogenicdifferentiation at different time points, and visualized undera Leica inverted microscope as shown in Fig. 4. During osteogenicdifferentiation, results suggest expression of osterix was evidentby day 5 of osteogenic differentiation, while osterix, osteocalcin

and collagen type I, were expressed by day 29 of differentiation.

Fig. 3. Progression of osteogenic and chondrogenic differentiation for murine embryonic stem cells induced to differentiate in osteogenic and chondrogenic media on CultiSpher Smicrocarriers. Photomicrographs represent day 0 (top row), day 10 (middle row) and day 30 (bottom row), murine ESCs induction to osteoblasts or chondrocytes. Cultures were

induced into osteogenic differentiation as a single cell inoculum with microcarriers (SC MCs) agitated at 60 rpm (a, f & k), as a cell-loaded inoculum (CL MCs) agitated at 60 rpm

(b, g & l) or as a cell-loaded inoculum (CL MCs) agitated at 100 rpm (c, h & m). Chondrogenic-differentiated cultures were induced either as a cell-loaded inoculum (CL MCs)

agitated at 100 rpm (d, i & n) or at 60 rpm (e, j & o). Fluorescent Activated Cell Sorting Analyses (FACS) for days 5, 14 and 29 osteoblasts and chondrocytes, harvested from CultiSpher

S microcarriers, indicate over 70% expression of markers for osteogenic and chondrogenic differentiation including osterix, collagen I and osteocalcin in all osteogenic culture

conditions (p, b & r), while aggrecan and collagen II and were expressed in chondrogenic cultures as shown in (s & t). Graphs represent days 5, 14 and 29 progression of osterix (p),

collagen type 1 (q), osteocalcin (r) osteogenic-differentiated cells. Expression of aggrecan (s) and collagen type II (t) were also assessed in chondrogenic-differentiated cells agitated

at 60 or 100 rpm.



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Expression of aggrecan and collagen type II, markers, which areindicative of a fully mature state of chondrogenesis, were observedby day 14 of differentiation. Similar results were observed whenosteogenic and chondrogenic cell-loaded microcarrier cultureswere dissociated and immunocytochemistry performed on disso-ciated cell suspension as shown in Fig. 4 (gei).

3.4. Histological analyses of cell-loaded CultiSpher S microcarrier

culture

von Kossa and Alizarin Red S analyses of induced osteoblastscells (Fig. 5) revealed that mineralization had occurred for all theconditions tested including; cell-loaded osteogenic inducedmicrocarriers (CL MCs) agitated at 100 rpm (Fig. 5 (a & e)), cell-loaded osteogenic induced microcarriers (CL MCs) agitated at60 rpm (Fig. 5 (b & f)) and single cell plus microcarrier (SC MC)osteogenic induction, agitated at 60 rpm (Fig. 5 (c & g)). A quali-tative comparison of mineralization by von Kossa and Alizarin Red Shistological analyses for calcium deposition, suggests no difference

in the levels of differentiation between both inoculum forms.

Positive controls forvon Kossa and Alizarin Red S, shown in Fig. 5 (d& h), respectively, were bovine bone tissue samples.

Histological analyses were also performed on chondrogenic-differentiated samples for the presence of GAGs by staining withalcian blue. Day 29 chondrogenic-differentiated cells showedevidence of cartilage formation, staining positive for alcian blue asshown in Fig. 5 (i & j) for cell-loaded chondrocytes agitated at100 rpm and 60 rpm, respectively. Bovine articular cartilage tissuewas used as positive controls (5 l), while negative controls (osteo-genic cell-loaded microcarriers) showed no evidence of GAGs, (5 k).A qualitative comparison of chondrogenic-differentiated culturesagitated at 100 rpm compared to cultures at agitated at 60 rpm,showed no difference in the level of alcian blue expressed withinsamples as seen in Fig. 5 (i vs. j).

3.5. Histological analyses of excised tissue after ectopic

implantation

To determine osteogenic potential in vivo, aggregates of osteo-

blasts were harvested on day 15 from the bioreactors inoculated

Fig. 4. Immunocytochemical analyses for bioreactor ESC-derived osteoblasts and chondrocytes. Photomicrographs represent brightfield and fluorescent images of intact cell-loadedmicrocarriers (CL MCs) analyzed for markers of osteogenic induction including osterix, osteocalcin and collagen type I (aef). Suspended osteoblasts cells were also harvested from

cell-loaded microcarriers (gei) and immunoassayed for markers of osteogenic differentiation including osterix (g), osteocalcin (h) and collagen type I (i). Immunocytochemistry was

also performed on days 5, 14 and 29 whole intact chondrogenic aggregates, and stained with markers for chondrogenic differentiation including aggrecan and collagen type II.

Suspended chondrogenic cells were also harvested from microcarriers and immunoassayed for markers of chondrogenic differentiation including aggrecan (p) and collagen type II

(g). Control samples (r) were stained only with secondary antibodies. For immunocytochemical analyses, intact cell-loaded microcarriers were cultivated in bioreactors, inoculated

at 60,000 cells/mL and agitated at 60 rpm. For harvested and dissociated cultures, suspended cells were seeded into 24 well plates at a density of 60,000 cells/mL in differentiation

medium and incubated at 37 and 5% CO2. Cultures were analyzed after 48 h. Scale bars represent 200 mm.



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with single cells without microcarriers (SC-MC) and implantedsubcutaneously into SCID mice. Ectopic implantation was alsoperformed on a separate set of mice using day 15 cell-loadedmicrocarriers (CL MCs) undergoing osteogenic differentiation.After 6e8 weeks the animals were euthanized and tissue at thepoint of injection was dissected and preserved in 10% formalin forhistology. Tissue samples were excised from the site of implanta-tion and analyzed as shown in Fig. 6. Mineralization was evidencedby black mineral deposition in tissue samples stained with von

Kossa (6b), while red nodule formation was visible in tissuesamples stained with Alizarin red S (6c).Histological analyses were also performed on tissue samples

using Massons Trichrome and H&E staining to determine the typesof tissue present in the excised tissue samples. Massons Trichromeproduces a red coloration for keratin and muscle fibers and a blueor green coloration for collagen and bone. Analyses of samplestransplanted with osteoblasts aggregates cultured without micro-carriers (SC-MCs), revealed muscle fiber tissue formation primarilyby day 15 of differentiation, with little collagen deposition asshown in Fig. 6d. Tissue samples harvested from SCID miceimplanted with cell-loaded microcarriers (CL MCs) showedevidence of collagen deposition by the cells from 6 weeks, with theeffects becoming more noticeable after 8 weeks of transplantation

as seen in Fig. 6 (e & f).

Finally, Hematoxylin and eosin (H&E) analyses of excised tissuerevealed formation of muscle tissue, 8 weeks after implantation ofosteogenic aggregates as shown in Fig. 6 g. However, when cellswere expanded, differentiated, and then transplanted into SCIDmice while still on microcarriers (SC MCs or CL MCs), H&Etissue analyses revealed formation of bone tissue in excised tissue.Bone tissue formation was evident after 6 weeks when the firsttissue was excised and was more evident after 8 weeks of trans-plantation as shown in Fig. 6 (h & i) respectively.

3.6. Orthotopic implantation of differentiated cells into burr-hole

fractures

For this study, the burr-hole fracture model previously reportedby Taiani et al. [39] was generated in five 8-week old femaleovariectomized Sv129 mice (the mice were strain-matched to theD3 ESCs used in this study). Day 25 cell-loaded CultiSpher Smicrocarriers (CLMCs) were used for implantation into the burr-hole fractures. Micro-CT imaging performed at Day 0, and at Weeks1, 2 and 4 post-implantation showed formation of new cortical andtrabecular bone at the fracture site as seen in Fig. 7 (aed). Impor-tantly, no abnormal disruption of the bone architecture, which

could indicate tumor formation, was detected in any of the 5 mice

Fig. 5. von Kossa (top row) and alizarin red S (middle row) histological analyses of cell-loaded microcarrier cultures show mineral deposition (black deposits) in osteogenic cultures.

Photomicrographs represent von Kossa and alizarin Red S analyses of: osteoblasts cell-loaded microcarriers, agitated at 100 rpm (a & e) respectively; osteoblasts cell-loadedmicrocarriers, agitated at 60 rpm (b & f) respectively, and single cell osteogenic induced microcarriers, agitated at 60 rpm (c & g) respectively. Positive controls for von Kossa

and alizarin red (d & h). Bottom row represents chondrogenesis in cell-loaded microcarrier (CL MC) cultures agitated at 100 rpm (i) or at 60 rpm (j). Tissue formation indicated by

dark blue coloration in chondrogenic cultures. Positive controls for alcian blue is represented in (l) while (k) represents negative controls for alcian blue. Scale bars represent

200 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).



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for up to 4 weeks after implantation of the cells. Furthermore, newcortical bone had bridged the burr-hole defect in all mice.

4. Discussion

Though the importance of developing effective systems to scale-up production of osteoblasts and chondrocytes to meet the risingdemand of these cells for cell therapy has been realized for a while,thus far, only a few studies have explored the use of bioreactors forthe tissue engineering of bone constructs from human bonemarrow stromal cells [43,44]. Others have investigated murineembryonic stem cell encapsulation and differentiation to osteo-blasts and chondrocytes [15,45e50]. Investigations have also beenperformed on endochondral ossification of cartilage formed byESCs seeded on scaffolds [51], while others have exploredproduction of tissue-engineered cartilage with and without EBformation [52,53]. This is the first time however, that mESCs havebeen differentiated to osteoblasts and chondrocyteson CultiSpher Smicrocarriers in bioreactors under feeder-free and serum-free

conditions.

In the current study, stirred suspension bioreactors provided aneffective controllable method for differentiating ESCs to osteoblastsand chondrocytes on a biodegradable, macroporous, collagen-basedmicrocarrier. CultiSpher S microcarriers offer several advantagesover other microcarrier types including effective harvesting, shear

protection and biodegradability. Unlike other types of microcarriersthat areprimarily glass or plastic, usuallycoated, CultiSpher S beadsare made up of a highly cross-linked type A porcine-derived gelatinmatrix. The ability to effectively harvest cells from microcarriers isan important aspect of microcarrier cell cultures especially duringscale-up. The macroporosity and gelatinous composition of CultiS-pherSbeadsenableseasycellharvestasthemicrocarriercompletelydisintegrates, leaving behind a single cell suspension. Histologicalanalysis was also possible as a result of easy sectioning of thesamples. In addition, CultiSpher S beads are FDA approved and haverecently been used for in vivo dermis regeneration in humans[54,55]. Therefore, CultiSpher S microcarriers are a promising scaf-fold for use in the production of murine ESC-derived cells andeventual implantation of derived cells for the treatment of degen-

erative diseases.

Fig. 6. Sample tissue excised from mice 8 weeks after subcutaneous implantation (a). von Kossa and alizarin Red S analyses of excised tissue samples from SCID mice after 8 weeks

of transplantation (b & c) respectively, show mineral deposition within tissue samples. Masson s Trichrome histological analyses of excised tissue show evidence of bone formation

in osteogenic tissue. Photomicrographs represent excised tissue, 8 weeks after implantation with osteogenic aggregates, without microcarriers (d), excised tissue, 6 and 8 weeks

after implantation with osteoblasts cell-loaded microcarriers (e & f) respectively. Bottom row represents H&E histological analyses of excised tissue 8 weeks after transplantation

with osteogenic aggregates without microcarriers (g), 6 and 8 weeks after transplantation with osteoblasts cell-loaded microcarriers (h & i). Aggregate cultures without micro-

carriers were inoculated at 60,000 cells/mL while microcarrier cultures were inoculated at 4000 cells/mL and incubated for 30 days at 37 C and 5% CO2. Black arrows indicate

collagen deposition and bone formation, while green arrows indicate muscle fibers. Scale bars represent 200 mm. (For interpretation of the references to colour in this figure legend,

the reader is referred to the web version of this article).



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When the effect of inoculum form (single cells microcarriersversus cell-loaded microcarriers) on differentiation was assessed,immunocytochemical analyses as well as histological analysesshowed mineralization could be observed under all conditions. Thisis important especially for cell cultures that require passaging asaggregates, as CultiSpher microcarriers may be passaged aftercomplete, partial or no dissociation at all. Alizarin red staining was

used to examine the presence of calcium deposition [56], as thepresence of phosphate (as determined by von Kossa) does notnecessarily imply that calcium is present in tissue [38]. Further,when twodifferenthydrodynamic shear forces (60 versus 100 rpm)were applied to the cultures, von Kossa and alizarin red S analysesof cultures suggests there were no differences in differentiationefficiencies between the cultures. The size of clumps was alsocontrolled with increased shear, which is in agreement withprevious studies that have found that agitation rates could be usedto control the size of ESC aggregates in bioreactor cultures[42,57,58]. Overall, thesefindings suggest that ESCs may be inducedinto osteogenic differentiation with the same efficacy whencultures are differentiated as single cells inoculated onto micro-carriers (SC MCs) or when differentiation is initiated on previ-

ously colonized microcarriers (CL MCs). Differentiation onCultiSpher S microcarriers led to cells retaining a high expression(>70%) of osteogenic markers in serum-free medium. In addition,higher agitation rates resulted in better control of cellularoutgrowths in cultures. CultiSpher S microcarriers presents a greatadvantage for cultivation of cell cultures at high agitation rates(>100 rpm), as their macroporous configuration protects cellsagainst shear damage. This is a desirable characteristic for scaffoldsused in differentiating cell cultures, particularly in agitatedcultures, as previous studies of ESC differentiation have found thathydrodynamic shear can lead to the maintenance of pluripotency[39,59]. In addition, the configuration of cells grown in CultiSpher Smiocrocarriers allows for effective differentiation without theformation of layers that have plagued osteogenic differentiation of

ESCs as micromass aggregates in suspension bioreactors [59]. The

study by Yamashita (2010) found that cell culture methods hadsignificant influence on ESC differentiation to osteoblasts. Histo-logical analyses of osteogenic aggregates produced by micromasscultivation in the study revealed there was an inner core ofmineralized cells that were surrounded by a layer of non- miner-alized cells lying underneath a layer of extracellular matrix. The useof CultiSpher S microcarriers with average pore diameters of about

20 mm, may prevent development of these layers and henceenhance bone regeneration in vivo.

Though the above described methodologies were effective atdifferentiating ESCs to osteoblasts and chondrocytes, the efficien-cies of differentiation as quantified by flow cytometry analyses formarkers of differentiation was only 80% at best. This was found tobe the case for all cultures tested. It may be possible to improvedifferentiation outcomes by investigating the use of different typesof bioreactors or improving the scaffold design to better enhancedifferentiation [60e62]. However, incorporating sorting tech-niques, including sorting by fluorescent activated cell sorting(FACS) or magnetic activated cell sorting (MACS), may furtherpurify cultures prior to transplantation, or select for a population ofprogenitors which will enhance differentiation efficiencies.

When tissue samples were harvested 6 and 8 weeks afterectopic implantation of osteoblast cell-loaded CultiSpher S micro-carriers into SCID mice and the results compared to tissue samplesimplanted with osteogenic aggregates only, results showedevidence of collagen deposition by the cell-loaded microcarriers asearly as 6 weeks which became more evident after 8 weeks ofimplantation. However, analyses of samples transplanted withosteoblast aggregates only revealed muscle fiber tissue formationeven 8 weeks after transplantation. This suggests that formation ofbone tissue was enhanced with the use of a collagen-based scaffold.

Orthotopic implantation of cell-loaded microcarriers intoa burr-hole fracture resulted in the formation of new bone at thefracture site. Although this type of fracture has been shown to healwithout therapeutic intervention [39], it is important to note that

no abnormal disruption of the bone architecture was observed

Fig. 7. Representative micro-CT images of orthotopic implantation of day 25 osteoblast-loaded microcarriers in a burr-hole fracture in one of the five mice. Images represent day

0 (a), week 1, (b), week 2 (c) and week 4 (d) from the same mouse.



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following implantation of the osteoblast-loaded CultiSpher Smicrocarriers. The formation of large soft tissue masses that causedisruption of the bone architecture has been observed by Taianiet al. (2011, submitted) following the implantation of some types ofESC-derived skeletal cells into the burr-hole fracture. No suchdisruptions were observed in the current study, and in contrast,fractures were observed to heal normally. This is a significantfinding, particularly in the field of regenerative medicine usingembryonic stem cells. Over the past decade, there has beensignificant skepticism regarding the case for using ESC-derived cellsfor tissue engineering and regenerative medicine applicationsbecause of the issue with tumor formation upon implantation. Onepossible explanation for this is the fact that the development oftumors appears to be linked to the use of animal based serum in thecell culture media. Implantation of ESC-derived cells cultured inbioreactors in serum-containing medium and later implantationinto a burr-hole defect in bone has been shown to result in tumorformation. However, when the cells were implanted after differ-entiation in static tissue culture, no tumors were observed [39]. Inthe current study, we have demonstrated that the elimination ofanimal based serum in culture media may inhibit formation oftumors post-implantation.

Finally, human embryonic stem cells (hESCs) are generallypassaged in small clumps, as complete dissociation of hESCs duringpassaging has been shown to result in cell death [40,63]. The use ofCultiSpher S microcarriers presents a great advantage for eventualhuman applications as these microcarriers could be dissociated intosmall clumps using collagenase, without complete dissociation intosingle cells. Overall, cultivation of ESCs on CultiSpher S micro-carriers in suspension bioreactors would allow for ESC-derivedcells to be produced in a robust and controllable environment,while protecting the cells from hydrodynamic shear.

5. Conclusions

Here, we have shown successful production of ESC-derivedosteoblasts and chondrocytes in suspension bioreactors. Resultssuggest effective differentiation to osteoblasts and chondrocytes ona macroporous gelatin based microcarrier. The biodegradablenature of CultiSpher S offers great potential for tissue engineeringbone and cartilage as it provides a suitable environment forexpansion and differentiation of ESCs and degrades upon trans-plantation, leaving only regenerated tissue where there was oncean injury. When the effect of inoculum form (single cell versus cell-loaded microcarrier inoculum) on the efficiency of differentiationwas investigated, no differences were observed. In addition,differentiation was effective at various agitation rates. Finally,orthotopic transplantation of derived cells did not result in tumorformation.

The methodologies developed in this research may provide

a path forward in the generation and transplantation of relevantnumbers of cells for use in musculoskeletal regenerative therapies.

Acknowledgments

RMA was awarded scholarships from the Province of Alberta(Queen Elizabeth II Scholarship). RMA, JTT and RK received fundingfrom the Skeletal Regenerative Medicine Team funded by the Cana-dian Institutes of Health Research (CIHR). RMA and JTT receivedfunding from the Biomedical Engineering Graduate Program at theUniversityof Calgary. JTT was funded through NSERC and AHFMR. AYwas supported by AHFMR and JSPS Fellowships. MSK and DERreceived funding through an NSERC Collaborative Health ResearchProject, and DER is an AHFMR Senior Scholar. We would like to thank

Dr JohnMatyasfrom the Facultyof Veterinary Medicine, UniversityofCalgary, Calgary, AB, Canada, T2N 4N1 for use of the ConfocalMicroscope and Maureen Bukhari from the University of CalgaryFaculty of Veterinary medicine for the histopathology work per-formed on specimens.

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