Biotechnol. Appl. Biochem. (2010) 55, 199–208 (Printed in Great Britain) doi:10.1042/BA20090356 199 Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll gradient Ana-Maria Rosca and Alexandrina Burlacu 1 Laboratory for Adult and Embryonic Stem Cell Biology, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, Hasdeu Street, Bucharest 050568, Romania Given the complex composition of bone marrow, a cell separation technique that results in populations enriched in progenitor cells is required for cellular dif- ferentiation and transplantation studies. In the present study, we designed a method that allows for the isolation of a progenitor-enriched population of bone marrow by exploiting the physical properties of these cells. Bone marrow aspirate was separated on a discon- tinuous Percoll gradient (ranging from 1.050 to 1.083 g/cm 3 ) that resulted in the recovery of six cell fractions. The fractions were characterized by FACS and RT–PCR (reverse transcription–PCR) analyses and evaluated for their capacity to differentiate into haematopoietic and mesenchymal cells. Fraction IV, including cells with a density of 1.070–1.076 g/ml, contained 11.68% of total bone marrow cells and was enriched in c-kit + and Sca-1 + (stem cell antigen-1) progenitor cells as compared with total bone marrow. This fraction demonstrated an increase in clonogenic capacity under specific conditions as well as a potential to generate a mesenchymal stem cell culture in a shorter period than that using bone marrow aspirate. Furthermore, this fraction lacked differentiated cell types and contained cells positive for endothelial markers, which further increases its value in cellular transplant. In conclusion, a bone marrow subpopulation that is enriched in progenitor cells and may be valuable in cellular transplant therapy can be isolated by exploiting the physical properties of these cells. Introduction Adult bone marrow is still recognized as the main source of stem/progenitor cells throughout the life of an adult. This tissue houses two stem cell populations with distinct progenies that are valuable in cellular transplant: HSCs (haematopoietic stem cells) and MSCs (mesenchymal stem cells) [1]. HSCs can give rise to all blood cells, whereas MSCs have been shown to differentiate in vitro, not only into mesenchymal lineages, such as osteoblasts, adipocytes and chondrocytes [1,2], but also into neurons and muscle [3]. However, stem/progenitor cells represent rare events among bone marrow nucleated cells [4,5]. They exist in a highly organized microenvironment composed of stromal cells (osteoblasts, adipocytes, endothelial cells and vascular pericytes), an extracellular matrix rich in fibronectin, collagens and proteoglycans [6] and a large amount of haematopoietic cells. Given the complex composition of bone marrow, a cell separation technique that results in subpopulations enriched in stem/progenitor cells is required for cellular differentiation and transplantation studies. At present, density gradient centrifugation using Histopaque is employed by many clinical investigators to separate progenitor cells from blood; however, it provides a pool of mononuclear cells containing all cells with a density of less than 1.077 g/ml, but no substantial improvement in progenitors. Other general methods to isolate progenitor cells from the mononuclear cell population include immunological methods [7], which are time-consuming and expensive because of the use of monoclonal antibodies and advanced technology. In the present study, we aimed to find out whether a bone marrow subpopulation enriched in stem/progenitor cells can be obtained by exploiting the physical properties of these cells. For this purpose, we designed a method to fractionate bone marrow cells, aiming in particular for cells with a density of less than 1.077 g/ml. The bone marrow suspension was layered on a discontinuous Percoll gradient, which allowed the fractionation of cells with a density of less than 1.077 g/ml into five subpopulations. Cells with a density higher than 1.077 g/ml remained at the bottom of the gradient in a distinct fraction. We showed that a subpopulation of bone marrow, which was enriched in small, c-kit + and Sca-1 + (stem cell antigen-1) progenitor Key words: bone marrow, c-kit, haematopoietic stem cell (HSC), mesenchymal stem cell (MSC), Percoll, stem cell antigen-1 (Sca-1). Abbreviations used: CFU, colony forming units; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HSC, haematopoietic stem cell; MSC, mesenchymal stem cell; PBS-FBS, PBS containing 2% FBS; PECAM, platelet endothelial cell adhesion molecule; RT–PCR, reverse transcription–PCR; Sca-1, stem cell antigen-1; SSC, side scatter; VEGFR2, vascular endothelial growth factor receptor 2. 1 To whom correspondence should be addressed (email [email protected]). C 2010 Portland Press Limited
Transcript
Biotechnol. Appl. Biochem. (2010) 55, 199–208 (Printed in Great Britain) doi:10.1042/BA20090356 199
Isolation of a mouse bone marrow population enriched in stemand progenitor cells by centrifugation on a Percoll gradient
Ana-Maria Rosca and Alexandrina Burlacu1
Laboratory for Adult and Embryonic Stem Cell Biology, Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’, HasdeuStreet, Bucharest 050568, Romania
Given the complex composition of bone marrow, acell separation technique that results in populationsenriched in progenitor cells is required for cellular dif-ferentiation and transplantation studies. In the presentstudy, we designed a method that allows for theisolation of a progenitor-enriched population of bonemarrow by exploiting the physical properties of thesecells. Bone marrow aspirate was separated on a discon-tinuous Percoll gradient (ranging from 1.050 to 1.083g/cm3) that resulted in the recovery of six cell fractions.The fractions were characterized by FACS and RT–PCR(reverse transcription–PCR) analyses and evaluated fortheir capacity to differentiate into haematopoietic andmesenchymal cells. Fraction IV, including cells witha density of 1.070–1.076 g/ml, contained 11.68% oftotal bone marrow cells and was enriched in c-kit+
and Sca-1+ (stem cell antigen-1) progenitor cells ascompared with total bone marrow. This fractiondemonstrated an increase in clonogenic capacity underspecific conditions as well as a potential to generate amesenchymal stem cell culture in a shorter period thanthat using bone marrow aspirate. Furthermore, thisfraction lacked differentiated cell types and containedcells positive for endothelial markers, which furtherincreases its value in cellular transplant. In conclusion,a bone marrow subpopulation that is enriched inprogenitor cells and may be valuable in cellulartransplant therapy can be isolated by exploiting thephysical properties of these cells.
Introduction
Adult bone marrow is still recognized as the main sourceof stem/progenitor cells throughout the life of an adult.This tissue houses two stem cell populations with distinctprogenies that are valuable in cellular transplant: HSCs(haematopoietic stem cells) and MSCs (mesenchymal stemcells) [1]. HSCs can give rise to all blood cells, whereasMSCs have been shown to differentiate in vitro, not onlyinto mesenchymal lineages, such as osteoblasts, adipocytesand chondrocytes [1,2], but also into neurons and muscle
[3]. However, stem/progenitor cells represent rare eventsamong bone marrow nucleated cells [4,5]. They exist in ahighly organized microenvironment composed of stromalcells (osteoblasts, adipocytes, endothelial cells and vascularpericytes), an extracellular matrix rich in fibronectin,collagens and proteoglycans [6] and a large amount ofhaematopoietic cells.
Given the complex composition of bone marrow, acell separation technique that results in subpopulationsenriched in stem/progenitor cells is required for cellulardifferentiation and transplantation studies. At present,density gradient centrifugation using Histopaque is employedby many clinical investigators to separate progenitor cellsfrom blood; however, it provides a pool of mononuclear cellscontaining all cells with a density of less than 1.077 g/ml, butno substantial improvement in progenitors. Other generalmethods to isolate progenitor cells from the mononuclearcell population include immunological methods [7], whichare time-consuming and expensive because of the use ofmonoclonal antibodies and advanced technology.
In the present study, we aimed to find out whethera bone marrow subpopulation enriched in stem/progenitorcells can be obtained by exploiting the physical propertiesof these cells. For this purpose, we designed a method tofractionate bone marrow cells, aiming in particular for cellswith a density of less than 1.077 g/ml. The bone marrowsuspension was layered on a discontinuous Percoll gradient,which allowed the fractionation of cells with a density ofless than 1.077 g/ml into five subpopulations. Cells witha density higher than 1.077 g/ml remained at the bottomof the gradient in a distinct fraction. We showed thata subpopulation of bone marrow, which was enriched insmall, c-kit+ and Sca-1+ (stem cell antigen-1) progenitor
cells, could be isolated by Percoll gradient centrifugation.This subpopulation, containing cells with a density varyingbetween 1.067 and 1.070 g/ml, had an increased capacityto generate haematopoietic cells under specific conditionsand, moreover, produced MSC cultures that were free ofhaematopoietic cells in a shorter period compared withthose from bone marrow aspirate.
Materials and methods
Isolation of bone marrow cellsAdult RAP mice (8 weeks old) were killed by cervicaldislocation in accordance with the rules of the InstitutionalEthical Board for Experimental Procedures. The tibiae andfemurs were harvested, and the medullar channels wereflushed with 5 ml PBS-FBS [PBS containing 2% FBS (fetalbovine serum)]. The single cell suspension obtained bymechanical dissociation (passing the cells through needlesof decreasing gauges, i.e. 20, 22 and 25 gauge) was thencentrifuged at 400 g for 5 min at 20 ◦C and resuspendedin 1 ml of DMEM (Dulbecco’s modified Eagle’s medium;Invitrogen). This volume of suspension was layered on thePercoll gradient or analysed by flow cytometry.
Separation on the Percoll gradientAn isotonic Percoll solution was made by mixing Percoll (25mOs/kg, 1.130 g/ml; Sigma–Aldrich) and 10× concentratedDMEM at a 9:1 volume ratio. Densities of 1.050, 1.057,1.067, 1.070, 1.076 and 1.083 g/ml were prepared by dilutingthe isotonic Percoll solution in DMEM at concentrationsof 35, 40, 45, 50, 55 and 60% respectively. A six-layeredPercoll gradient was obtained by layering 2 ml of eachPercoll solution. Finally, the cell suspension obtained asdescribed above was placed on the top of the gradient andcentrifuged at 1500 g for 25 min at 4 ◦C. Five fractions of2 ml each were collected after centrifugation and designatedI–V (see Figure 2A). The lower part of the gradient (3ml), which contained the cells with a density higher than1.076g/ml and included the erythrocytes, was collected asfraction VI. The cells in each fraction were washed threetimes in PBS-FBS and then either analysed or cultivatedin DMEM supplemented with 10% MSC-Qualified FBS(Invitrogen).
In situ detection of progenitor cellsTibiae were fixed in formalin and then decalcified in5% trichloroacetic acid, dehydrated in a graded seriesof alcohols and embedded in paraffin. Longitudinalsections (5 μm) on poly-L-lysine-coated glass slides weredeparaffinized and microwaved in citrate buffer for antigenretrieval. Anti-mouse c-kit or Sca-1 antibody (R&D Systems)
was used at 10 μg/ml. Incubation with the primary antibodywas followed by quenching in 3% H2O2 and incubationwith horseradish peroxidase-conjugated anti-mouse IgG.Ultimately, the proteins were visualized by the DAB(diaminobenzidine) reaction and examined under a Nikonmicroscope.
Flow cytometry assayFACS was performed using a MoFlo Cell Sorter. Cellsfrom fractions I–VI and bone marrow aspirate wereincubated with a phycoerythrin-labelled anti-c-kit or anti-Sca-1 antibody (R&D Systems) for 1 h at 4 ◦C, washed inPBS-FBS and analysed. In order to exclude dead cells fromthe analysis, 1 μg/ml propidium iodide was added just beforethe analysis. At least 20000 events were considered for eachsample. Results were analysed using Summit v4.3 software(Cytomation, Inc).
RT–PCR (reverse transcription–PCR)Total RNA was extracted using the RNeasy Micro kit (Qia-gen), and cDNA was synthesized from 0.2 μg of total RNAemploying MMLV Reverse transcriptase (Invitrogen). PCRwas performed using optimized amplification conditions, andthe products were visualized on a 1.5% agarose gel withethidium bromide staining. For each gene, DNA primerswere derived from different exons whenever possible, andcDNAs were treated with DNase I to ensure that the PCRproduct represented the specific messenger RNA speciesand not genomic DNA. The primer sequences are availableupon request.
Colony-forming unit assayThe functionality of the haematopoietic stem/progenitorcells within each fraction was estimated in HSC-CFUcomplete with Epo medium (Miltenyi Biotec). This mediumallows the enumeration of haematopoietic stem andprogenitor cells characterized as CFU (colony forming units)and has been reported to support the growth of granulocyte(CFU-G), macrophage (CFU-M), granulocyte/macrophage(CFU-GM) and erythroid [BFU-E (erythroid burst-formingunits) and CFU-E] colonies, as well as mixed colonies (CFU-GEMM) [8]. Cells were cultivated at 1500 cells/cm2 induplicates and clusters of cells were scored after 7 days.
Adipogenic and osteogenic differentiationCells from each fraction were seeded into two wells of a24-well plate. After 1 week in culture, the cells wereincubated for an additional week in either adipogenic[DMEM with 10% FBS, 10−6 M dexamethasone, 100 μMindomethancin and 1% ITS (insulin-transferrin-sodiumselenite supplement)] [9] or osteogenic (DMEM with 10%
Isolation of a progenitor-enriched population of bone marrow 201
Figure 1 Quantification and localization of c-kit+ and Sca-1+ cells in bone marrow
(A) Flow-cytometry quantification of c-kit+ and Sca-1+ cells in bone marrow. C-kit was identified on 11.8 +− 2.5% of the bone marrow cells with varying granularity,whereas Sca-1+ cells represented 9.8 +− 2.8% of bone marrow cells and were almost exclusively characterized by low granularity. Diagrams are representatives offour independent experiments, whose mean values are illustrated in the adjoined table. (B) Immunohistochemistry for in situ localization of c-kit+ (b and c) andSca-1+ (d, e and f) cells within bone marrow. Staining was completely absent in tissue sections in which the primary antibody was omitted (a). One can notice theepiphysis as preferential site for Sca-1+ cells (d–f), and the diaphysis for c-kit+ cells (b and c).
FBS, 10−7 M dexamethasone, 10 mM β-glycerophosphateand 0.3 mM ascorbic acid) [6] differentiation medium.All chemicals were from Sigma, unless otherwise stated.Lipid droplets were stained with Oil Red (Sigma), andlipid accumulation was quantified as previously described[10] using the TECAN iGenios spectrofluorimeter. Calciumdeposits were stained by the von Kossa method [6].
StatisticsAll quantitative results were expressed as themeans +− S.E.M. from at least three experiments. Statisticalanalysis was performed using one-way ANOVA. A p-valueless than 0.05 was considered significant.
Results
Localization and quantification of c-kit- andSca-1-positive cells in mouse bone marrowQuantification of progenitor cells from the bone marrowaspirate was determined on the basis of c-kit and Sca-1surface marker expression. Flow cytometry results revealedthat 11.8% of total bone marrow cells were c-kit+ cellsand 9.8% were Sca-1+ cells (Figure 1A). To localize theirpreferential site within bone marrow, longitudinal sectionsof decalcified tibiae were analysed by immunohistochemistry(Figure 1B). c-kit+ cells were predominantly localized inthe diaphyseal region (Figure 1B, b and c), whereas Sca-1+ cells were detected within the epiphyseal region of
Figure 2 Separation of bone marrow cells into six fractions by centrifugation on a discontinuous Percoll gradient
(A) Schematic illustration of the discontinuous Percoll gradient used for fractionation and the percentage of cells segregated in each fraction after centrifugation.(B) Dot-plot histograms resulting from FACS analysis illustrating the heterogeneity of cells before separation and the morphology of cells in each fraction.
the bone (Figure 1B, d–f). These results indicate thatcolonies of progenitor cells can be found in both theepiphysis and diaphysis, but differ in their expression of cellsurface markers. The segregation of the two populationsof progenitor cells in different sites within bone marrowruled out the existence of c-kit+/Sca-1+ double-positivecells in this tissue, indicating that the large majority ofprogenitor cells were either Sca-1 or c-kit single-positivecells. Our results corroborate other studies that revealedvery low percentages of Sca-1+/c-kit+ double-positive cellswithin bone marrow [11,12].
Separation of bone marrow cells on a PercollgradientBecause c-kit+ and Sca-1+ cells have a low level of granularity[as revealed by low SSC (side scatter), see Figure 1A], weconsidered the possibility of exploiting physical propertiesin order to separate these cells. Centrifugation of cells ona discontinuous Percoll gradient (ranging from 1.050 to1.083 g/cm3) resulted in the recovery of six cell fractions(I–VI), corresponding to the six densities of the gradient.The total number of cells recovered in the six fractionswas 35 × 106 +− 1.4 × 106 cells, meaning a recovery rateof 70% +− 2.8% (considering an average of 50 × 106 cells
harvested from one mouse). The segregation of cells withineach fraction is represented in Figure 2(A) as a percentageof the total amount of cells recovered after separation.The counting revealed that 0.58%, 0.62%, 1.73%, 11.68%,66.32% and 19.08% of cells were segregated in fractionsI–VI respectively, meaning 2 × 105 cells in fraction I, 2.2 × 105
in fraction II, 4.2 × 105 in fraction III, 4.3 × 106 in fractionIV, 23.7 × 106 in fraction V, and 6.8 × 106 in fraction VI.Erythrocytes residing mainly in fraction VI were excludedfrom the counting. Thus the majority of cells (66.32%)segregated in fraction V, corresponding to the densityrange 1.070–1.076 g/ml, whereas fraction IV containedonly a minor population of cells (11.68%). As expected,flow cytometry analysis of each subpopulation revealed anincrease in cell granularity [FSC (forward scatter)/SSC] withincreasing Percoll density (Figure 2B).
Quantification of the progenitor cells withinfractionsCells within each fraction were analysed for the presence ofc-kit+ and Sca-1+ progenitor cell markers by flow cytometry.As shown in Figure 3(A), the distributions of both c-kit+ andSca-1+ progenitor cells were confined to fractions III–V andwere not present in fractions I, II and VI. The quantification
Isolation of a progenitor-enriched population of bone marrow 203
Figure 3 Depiction of c-kit+ and Sca-1+ cells within fractions
(A) Flow cytometry diagrams revealing the percentage of c-kit+ and Sca-1+ cells within each fraction obtained after Percoll gradient separation of bone marrowaspirate. The progenitor cells are mainly found in fractions III, IV and V. The diagram is representative of three experiments, and the average percentages are givenin the bar charts below. (B) The overall representation of flow cytometry data showing the enrichment factor for c-kit+ and Sca-1+ cells in the six fractions relativeto bone marrow aspirate (unfractionated cells). The values denote the average of three independent experiments after extraction of the unspecific binding values.Note that fractions III and IV show the greatest enrichment for progenitor cells, illustrating 2.7- and 2.4-fold for c-kit+ cells and 1.9- and 2.2-fold for Sca-1+ cells, infraction III and IV respectively.
of these cells is given in Figure 3(A) as purity of each fractionand in Figure 3(B) as enrichment factor for c-kit+ cells (left)and Sca-1+ cells (right) as compared with bone marrow
aspirate. Analysis of fraction V, which contained the majorityof the cells (66.32% of the total bone marrow cells; seeFigure 2A), revealed no significant increase in the percentage
Figure 4 Evaluation of the clonogenic potential of the haematopoietic cells within the fractions
(A) Evaluation of differentiation potential within each fraction using the CFU assay. The diagram illustrates the average value of three independent experiments.Note that the highest capacity of differentiation resides in fractions III and IV (*P< 0.05). A representative field of fraction IV was photographed and displayedabove the diagram. (B) RT–PCR illustrating the presence of representative cell types within each fraction immediately after separation (left panel) and after twoweeks in culture (right panel). As a control, c-kit+ and Sca-1+ cell populations isolated by the FACS method are given in the middle panel. (C) Lower panel:spectrophotometric quantification of lipid accumulation in the six fractions after culturing the cells in adipogenic medium. The upper panel depicts a representativepicture of Oil Red staining in fractions III and IV. Right, light microscopy of von Kossa staining in the six fractions after cultivation in osteogenic medium. Note thanfractions III and IV, but not fraction V (that contained the majority of the cells), were able to generate osteoblasts in appropriate conditions.
of progenitor cells. Fraction V contained 14 +− 1.6% c-kit+
and 8 +− 4.6% Sca-1+ cells as compared with 11.8 +− 2.5%c-kit+ and 9.8 +− 2.8% Sca-1+ cells in the total bone marrowcell population. In contrast, fractions III and IV containedimportant enrichments for progenitor cells (either c-kit+ orSca-1+). Despite the enrichment factor of progenitor cells infraction III (2.74 +− 0.1 for c-kit+ cells and 1.9 +− 0.5 for Sca-1+
cells), the total amount of progenitor cells in fraction III wasinconsistent due to the low number of cells segregated inthis fraction (1.73%). Thus fraction III contained 1.8 × 105
progenitor cells out of 4.2 × 105 cells recovered in thisfraction.
In contrast, 23 +− 4.8% of cells in fraction IV werec-kit+ and an additional 22 +− 2.3% were Sca-1+ as comparedwith the unfractionated population (11.8% c-kit+ cells and9.8% Sca-1+ cells). These results suggested a 2.36 +− 0.7-fold
increase in c-kit+ cells and a 2.17 +− 0.3-fold increasein Sca-1+ cells in fraction IV as compared with theunfractionated bone marrow cell population. Thus fractionIV contained 4.3 × 106 cells, of which approx. 2 × 106 cellswere progenitors.
To evaluate the clonogenic potential of the haemato-poietic cells within fraction IV, the colony-forming assaywas performed on the cell fractions. To this purpose, 1.2 ×104 cells from each fraction were plated in methylcellulosemedium, and the cell clusters generated were scored after7 days in culture. As illustrated in Figure 4(A, upper panel),the colonies varied in size, probably reflecting the differencesin the proliferation rate and/or differentiation ability of thevarious progenitor cells within the fractions. Nevertheless,fractions III and IV generated the greatest number of CFU(Figure 4A). These fractions generated approx. 8.4 +− 2.3 and
Isolation of a progenitor-enriched population of bone marrow 205
7.8 +− 0.2 CFU per 103 cells, which was 2-fold higher thanthe major fraction, V (3.9 +− 1.9 CFU per 103 cells). Thisresult indicates enrichment in haematopoietic progenitorsin fractions III and IV. Conversely, fractions I, II and VIgenerated fewer CFU, as could be predicted from thedecreased number of c-kit+ cells and Sca-1+ cells scoredin those fractions.
Expression of differentiated cell markers in thefractionsTo better characterize the content of fraction IV, thedistribution of the main differentiated cell types that arenormally present within bone marrow was evaluated in eachfraction by RT–PCR and the results were compared withc-kit+ cells and Sca-1+ cells purified using FACS. Asillustrated in Figure 4(B, left panel), besides being enrichedin the expression of c-kit and Sca-1, fractions III and IVshowed the highest expression level of the endothelialmarkers VEGFR2 (vascular endothelial growth factorreceptor 2) and PECAM (platelet endothelial cell adhesionmolecule) of all six fractions. Furthermore, fraction IVdemonstrated a low level expression of differentiationmarkers of other cell types, such as adipocytes (fabp4),osteoblasts (osteocalcin) and chondrocytes (collagen),which segregated mainly within fractions I–III. As expected,the FACS method generated more purified cell populationsthat were devoid of other cell types.
After 2 weeks in culture, adherent cells were obtai-ned from all fractions (results not shown). Specifically,cells from fraction IV generated a culture of cells with afibroblast-like morphology that were distinct from thosederived from fractions III and V, mainly by lacking CD45mRNA expression (Figure 4B, right panel). Furthermore,adherent cells in fraction IV expressed Sca-1 and werenot contaminated with haematopoietic cells (as revealed bythe absence of CD45 and c-kit mRNA) or differentiatedcells, such as endothelial cells (PECAM/CD31 andVEGFR2/Flk-1), osteoblasts (osteocalcin) or chondrocytes(collagen II).
To evaluate which cell fractions could generateMSC cultures, the capacity of the cells to differentiateinto adipocytes and osteoblasts was evaluated for eachcell fraction after 1 week of cultivation. Although allfractions were able to generate adipocytes under theappropriate conditions, spectrophotometric quantificationof lipid accumulation (Figure 4C, lower panel) suggesteda greater capacity for fractions III–V as compared withthe other fractions. Nevertheless, the capacity to generateosteoblasts was limited to cells within fractions IIIand IV (Figure 4C, right panel), and fraction V lackeddifferentiated cells. These results suggest that fractions IIIand IV, but not fraction V, generated multipotent cells
during cultivation. Altogether, our results demonstrate thatfraction IV generated a cell culture that was depleted inhaematopoietic cells, negative for osteoblast, chondrocyteand endothelial markers, yet possessed a multipotentcapacity. These CD45−/c-kit−/Sca-1+/CD31−/Flk-1− cells aresimilar to bone marrow MSCs, which can be obtainedfrom bone marrow aspirate after several weeks in culture[13,14].
In conclusion, a Percoll gradient can be used toobtain a cell fraction that is enriched in progenitors andcan generate an MSC culture after a short period ofcultivation. This fraction is enriched in both haematopoieticand mesenchymal progenitor cells (c-kit+ and Sca-1+ cells),whereas it is deprived of other differentiated cell types, andalso contains cells that are positive for endothelial markers,which further increases its value in cellular transplant studieswhere angiogenesis is required.
Discussion
The existence of stem/progenitor cells within the hetero-geneous population of bone marrow is well documented[15]. Haematopoietic stem cells are the most primitivecells that retain multilineage haematopoietic differentiationpotential. In adult bone marrow, most haematopoieticprogenitors express c-kit (CD117), a member of a familyof cell-surface receptors with tyrosine kinase activitythat plays an important role in the regulation of theearly stages of haematopoietic development [16]. c-kit isexpressed in both long-term repopulating HSCs and moredifferentiated progenitor cells and is used as a valuablemarker for the identification of these cells [17]. Althoughc-kit is an established marker for HSCs, the expressionof Sca-1, a member of the Ly6 family, in these cells iscontroversial. To date, Sca-1 expression has been identifiedin putative stem/progenitor cell populations within varioustissues and organs including bone marrow, kidney, liver,lung, pancreas and muscle, as well as the aorta-gonad-mesonephros, yolk sack and other regions of the embryo[18]. However, whether these Sca-1+ populations are tissue-specific precursor/stem cells or represent haematopoietic,mesenchymal or endothelial precursor/stem cells associatedwith these tissues is not known in all cases [18].
We show in the present paper that c-kit+ and Sca-1+
cells segregated within bone marrow with respect tolocation, thus indicating that the large majority of progenitorcells were either Sca-1 or c-kit single-positive cells. Thiswas also strengthened by the RT–PCR analysis on FACS-separated c-kit+ and Sca-1+ populations that revealed theabsence of c-kit mRNA in a Sca-1+ population, even ifsome c-kit+ cells do express Sca-1 at the mRNA level(Figure 4B). In accordance with these results, very low
percentages of Sca-1+/c-kit+ double-positive cells withinbone marrow have previously been quantified by FACSanalysis [11,12]. For example, less than 2% out of 20%c-kit+ cell populations and 10% Sca-1+ cell populationsrespectively has been found to double-stain for c-kit andSca-1 in bone marrow mononuclear cells [12]. Anotherstudy reported that only 0.2% of Lin− cells in bone marrowwere Sca-1+/c-kit+ double-positive [11].
Because c-kit+ and Sca-1+ cells have low levelsof granularity, we considered the possibility of usingthis physical property in order to separate these cells.Our study showed that a bone marrow subpopulationthat is enriched in Sca-1+ and c-kit+ cells can beobtained through density gradient centrifugation and hasthe ability to differentiate under specific conditions. Thisfraction can be a source for MSCs (CD45−/c-kit−/Sca-1+/PECAM−/VEGFR2−/osteocalcin−/collagenII− cells) that canbe used in differentiation and cellular transplantation studies.
The advantage of using a Percoll gradient to enrichfor cell populationsCurrently, various techniques based on the physical andimmunochemical characteristics of stem/progenitor cells areused for their enrichment. Among these, FACS or magneticbead separation, which are based on cell surface antigenexpression, are most commonly used for either positive ornegative selection [19,20]. These immunochemical methodspresent the disadvantage of being time-consuming andexpensive because of the use of monoclonal antibodiesand advanced technology. Although more purity is givenby immunological assays in isolating cells that express acertain surface marker, the resulting cell populations arestill heterogeneous. That is because no single specificstem cell marker has been identified yet. Likewise, stemcells are known to express a wide variety of antigensexpressed by many other cell types, and these antigensare variably expressed depending on cellular status. Onthe other hand, mesenchymal stem cell cultures isolated byimmunodepletion of haematopoietic cells have been foundto exhibit poor growth mainly due to the absence of othercells that normally supported their growth by secretingparacrine factors. Furthermore, our trials to cultivate apure population of c-kit+ and Sca-1+ cells isolated by theFACS method also failed due to the absence of survivalfactors provided by supporting cells (results not shown).Thus physical separation of bone marrow cells remains agood alternative to immunochemical methods.
Since its introduction in 1977, Percoll has been usedto isolate several cell types. Its nearly ideal physicalcharacteristics make it especially useful as a first stepin the enrichment of cell populations before attemptinghigher resolution. With the use of this method, several
studies showed a successful enrichment of haematopoieticprogenitor cells that were able to repopulate lethallyirradiated mice. Nijhof and Wierenga isolated cells witha diameter between 7.2 and 8.4 μm, which sedimentat a density of 1.065 g/ml, from spleen; these cells hada high capacity to repopulate lethally irradiated micewith all haemopoietic precursor cells [21]. In accordancewith that study, our flow cytometry results showed apreferential distribution of c-kit and Sca-1 progenitorcell markers in a population characterized by a densitybetween 1.057 and 1.076 g/ml. By using a discontinuousgradient centrifugation, mouse bone marrow cells wereseparated into six fractions (corresponding to 1.050, 1.057,1.067, 1.070, 1.076 and 1.083 g/ml), and progenitor cellswere significantly enriched within fractions III–V. In anycase, fraction IV was particularly important because itharboured approx. 2 × 106 stem/progenitor cells out of atotal of 4.3 × 106 cells. This population is highly enrichedin stem/progenitor cells compared with the initial bonemarrow population and is valuable in various experiments,including cellular transplantation studies, which usually needbetween several thousand and two million cells.
Fraction IV as a source of HSCsThere is always a compromise in choosing the conditions forisolating HSCs between those that provide the highest yieldand those that provide the highest content of progenitorcells. As a matter of choice, two techniques, namelyelutriation and labelling with a selective progenitor marker,are being differentially used for this purpose.
The method described in the present paper provides analternative for the isolation of a haematopoietic-progenitor-cell-enriched population from bone marrow, in which thephysical properties of the progenitor cells are exploited. Thecells in fraction IV, even though they are still heterogeneous,contain a superior amount of haematopoietic progenitors,namely 45% of the population, and furthermore, a highamount of endothelial progenitor cells. Besides thesehaematopoietic progenitor cell populations, this fractionstill harbours several other cell types, among which arechondroblasts and adipocytes. As a proof of its improvementfor HSCs, this subpopulation is able to generate an increasednumber of haematopoietic colonies when cultivated underthe appropriate conditions. No prediction can be madeabout the short- or long-term reconstituting properties ofthese cells, as they are morphologically indistinguishableand such discrimination can be only based on in vivofunctional assays. It is likely that a number of cells havereconstituting abilities (as we know that such cells residewithin bone marrow); however, we did not perform in vivoexperiments and consequently we have no evidence on thisissue.
Isolation of a progenitor-enriched population of bone marrow 207
Fraction IV as a source of MSCsMSCs are usually isolated on the basis of their adherenceto plastic surfaces. Based on this property, MSCs fromdifferent species have been obtained and proven to beable to expand in culture while retaining their multipotentdifferentiation capacity. Mouse MSC cultures are frequentlycontaminated by haematopoietic cells; hence, these cells aremore difficult to isolate and expand as compared with MSCsfrom other species [13]. Several approaches for isolatingpure populations of mouse MSCs have been reported. Forinstance, immunodepletion of haematopoietic precursorshas been used as a first purification step of bone marrowaspirate in order to obtain a more homogenous cultureof MSCs [19,20]. However, the cells have been found toexhibit poor growth due to the dramatic down-regulationof many genes involved in cell proliferation and cell cycleprogression as a result of immunodepletion [19]. As analternative to immunodepletion, mouse MSC cultures canbe obtained from diverse strains by incubating total bonemarrow cells in a high-density culture with medium thatinhibits the growth of haematopoietic cells. In this approach,the cells are grown under the aforementioned conditionsfor the first two passages (5–6 weeks), after which the mosteasily detached cells are expanded in a low-density culture[21]. In another reported strategy, cells are plated at 106
cells/cm2 and the medium in the primary culture is changedfrequently concomitant with a diminished trypsinization time[22]. In this procedure, a purified population of MSCs canbe obtained 3 weeks after the initiation of culture (twopassages). In the method described in the present study, asimilar pure MSC population (containing CD45−/c-kit−/Sca-1+/PECAM−/VEGFR2−/osteocalcin−/collagenII− cells) can beobtained by cultivating cells from fraction IV for no morethan 1 week in DMEM supplemented with 10% MSC-qualified FBS.
In conclusion, we designed a method for thefractionation of mouse bone marrow through the useof a Percoll gradient that resulted in a progenitor-cell-enriched population. This population of small cellssegregated at a density of 1.070 g/ml and contained bothmesenchymal and haematopoietic progenitors. These cellswere able to generate more haematopoietic colonies underthe appropriate conditions as compared with total bonemarrow. They were also able to produce an MSC line thatwas free of haematopoetic cells in a short time in culture ascompared with classical methods.
Acknowledgements
We thank Eugen Andrei, Ioana Manolescu and Dr EmanuelDragan for their technical assistance.
Funding
This work was supported by grants from the RomanianMinistry of Education and Research, IDEA Program [grantnumber 250/2007] and COST (European Co-operation inScience and Technology) Action BM0602.
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