Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESCs) is a prerequisite for their use in clinical applications. However, there is no standard protocol for differentiating hESCs into osteoblastic cells. The aim of this study was to identify the emergence of a human stromal (mesenchymal and skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESCs in a feeder-free environment using serum replacement and as suspension aggregates (embryoid bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106, and CD166 as revealed by immunohistochemical staining and flow cytometry (fluorescence-activated cell sorting) analysis. Ex vivo differentiation of hEBs using bone morphogenic protein 2 (BMP2) combined with standard osteoblast induction medium led to weak osteoblastic induction. Conversely, subcutaneous in vivo implantation of day 20 hEBs in immune deficient mice, mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) as an osteoconductive scaffold, revealed bone and cartilage, and fibrous tissue elements after 8 weeks. These tissues were of human origin and there was no evidence of differentiation to nonmesodermal tissues. hEBs implanted in the absence of HA/TCP formed vacuolated tissue containing glandular, fibrous and muscle-like tissue elements. Conversely, implantation of undifferentiated hESCs resulted in the formation of a teratoma containing a mixture of endodermal, mesodermal, and ectodermal tissues. Our study demonstrates that hMSC-like cells can be obtained from hESCs and they can be induced to form skeletal tissues in vivo when combined with HA/TCP. These findings are relevant for tissue engineering and suggest that differentiated hEBs can provide an unlimited source for functional osteogenic cells.
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References
1.
AbdallahBM, KassemM. 2008. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther, 15:109–116.
2.
StenderupK, JustesenJ, ClausenC, KassemM. 2003. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone, 33:919–926.
3.
BarberiT, WillisLM, SocciND, StuderL. 2005. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Med, 2:e161.
4.
BarberiT, BradburyM, DincerZ, PanagiotakosG, SocciND, StuderL. 2007. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med, 13:642–648.
5.
LianQ, LyeE, Suan YeoK, Khia Way TanE, Salto-TellezM, LiuTM, PalanisamyN, El OakleyRM, LeeEH, LimB, LimSK. 2007. Derivation of Clinically Compliant MSCs from CD105+, CD24- Differentiated Human ESCs. Stem Cells, 25:425–436.
6.
TrivediP, HemattiP. 2007. Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells. Exp Hematol, 35:146–154.
7.
MahmoodA, HarknessL, SchroderHD, AbdallahBM, KassemM. 2010. Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/activin/nodal signaling using SB-431542. J Bone Miner Res, 25:1216–1233.
8.
HyslopLA, ArmstrongL, StojkovicM, LakoM. 2005. Human embryonic stem cells: biology and clinical implications. Expert Rev Mol Med, 7:1–21.
9.
Itskovitz-EldorJ, SchuldinerM, KarsentiD, EdenA, YanukaO, AmitM, SoreqH, BenvenistyN. 2000. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med, 6:88–95.
10.
CaiJ, ChenJ, LiuY, MiuraT, LuoY, LoringJF, FreedWJ, RaoMS, ZengX. 2006. Assessing self-renewal and differentiation in human embryonic stem cell lines. Stem Cells, 24:516–530.
11.
OlivierEN, RybickiAC, BouhassiraEE. 2006. Differentiation of human embryonic stem cells into bipotent mesenchymal stem cells. Stem Cells, 24:1914–1922.
12.
BielbyRC, BoccacciniAR, PolakJM, ButteryLDK. 2004. In Vitro Differentiation and In Vivo Mineralization of Osteogenic Cells Derived from Human Embryonic Stem Cells. Tissue Eng, 10:1518–1525.
13.
SottileV, SeuwenK. 2000. Bone morphogenetic protein-2 stimulates adipogenic differentiation of mesenchymal precursor cells in synergy with BRL 49653 (rosiglitazone)FEBS Lett, 475:201–204.
14.
CowanCA, KlimanskayaI, McMahonJ, AtienzaJ, WitmyerJ, ZuckerJP, WangS, MortonCC, McMahonAP, PowersD, MeltonDA. 2004. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med, 350:1353–1356.
15.
LarsenKH, FrederiksenCM, BurnsJS, AbdallahBM, KassemM. 2010. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacity. J Bone Miner Res, 25:796–808.
16.
SimonsenJL, RosadaC, SerakinciN, JustesenJ, StenderupK, RattanSI, JensenTG, KassemM. 2002. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol, 20:592–596.
17.
AbdallahBM, DitzelN, KassemM. 2008. Assessment of bone formation capacity using in vivo transplantation assays: procedure and tissue analysis. Methods Mol Biol, 455:89–100.
18.
AbdallahBM, KassemM. 2009. The use of mesenchymal (skeletal) stem cells for treatment of degenerative diseases: current status and future perspectives. J Cell Physiol, 218:9–12.
19.
GronthosS, ZannettinoAC, HaySJ, ShiS, GravesSE, KortesidisA, SimmonsPJ. 2003. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci, 116:1827–1835.
StenderupK, RosadaC, JustesenJ, Al-SoubkyT, gnaes-HansenF, KassemM. 2004. Aged human bone marrow stromal cells maintaining bone forming capacity in vivo evaluated using an improved method of visualization. Biogerontology, 5:107–118.
22.
CoucouvanisE, MartinGR. 1999. BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development, 126:535–546.
CoucouvanisE, MartinGR. 1995. Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell, 83:279–287.
25.
ConleyBJ, TrounsonAO, MollardR. 2004. Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives. Fetal Diagn Ther, 19:218–223.
26.
KellerG. 2005. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev, 19:1129–1155.
27.
HwangNS, VargheseS, LeeHJ, ZhangZ, YeZ, BaeJ, ChengL, ElisseeffJ. 2008. In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proc Natl Acad Sci U S A, 105:20641–20646.
28.
Olivares-NavarreteR, RainesAL, HyzySL, ParkJH, HuttonDL, CochranDL, BoyanBD, SchwartzZ. 2012. Osteoblast maturation and new bone formation in response to titanium implant surface features are reduced with age. J Bone Miner Res.[Epub ahead of print]10.1002/jbmr.1628.
29.
ButteryL, BielbyR, HowardD, ShakesheffK. 2011. Osteogenic differentiation of embryonic stem cells in 2D and 3D culture. Methods Mol Biol, 695:281–308.
30.
BoydNL, DharaSK, RekayaR, GodbeyEA, HasneenK, RaoRR, West FDIII, GerweBA, SticeSL. 2007. BMP4 Promotes Formation of Primitive Vascular Networks in Human Embryonic Stem Cell-Derived Embryoid Bodies. Exp Biol Med, 232:833–843.
31.
HarknessL, MahmoodA, DitzelN, AbdallahBM, NygaardJV, KassemM. 2011. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential. Bone, 48:231–241.
32.
UllmannU, In't VeldP, GillesC, SermonK, De RyckeM, Van de VeldeH, Van SteirteghemA, LiebaersI. 2007. Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Mol Hum Reprod, 13:21–32.
33.
KarpJM, FerreiraLS, KhademhosseiniA, KwonAH, YehJ, LangerRS. 2006. Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro. Stem Cells, 24:835–843.
34.
VodyanikMA, BorkJA, ThomsonJA, SlukvinII. 2005. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood, 105:617–626.
35.
InancB, ElcinAE, ElcinYM. 2007. Effect of osteogenic induction on the in vitro differentiation of human embryonic stem cells cocultured with periodontal ligament fibroblasts. Artif Organs, 31:792–800.
36.
KarnerE, UngerC, SloanAJ, hrlund-RichterL, SugarsRV, WendelM. 2007. Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro. Stem Cells Dev, 16:39–52.
37.
KarnerE, UngerC, CernyR, hrlund-RichterL, GanssB, DilberMS, WendelM. 2008. Differentiation of human embryonic stem cells into osteogenic or hematopoietic lineages: a dose-dependent effect of osterix over-expression. J Cell Physiol, 218:323–333.
38.
KawaguchiJ, MeePJ, SmithAG. 2005. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone, 36:758–769.
39.
FrandsenU, PornekiAD, FloridonC, AbdallahBM, KassemM. 2007. Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies. Biochem Biophys Res Commun, 362:568–574.
40.
KimSE, KimBK, GilJE, KimSK, KimJH. 2007. Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro. Mol Cells, 23:49–56.
41.
LaflammeMA, ChenKY, NaumovaAV, MuskheliV, FugateJA, DuprasSK, ReineckeH, XuC, HassanipourMet al.2007. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol, 25:1015–1024.
42.
SottileV, ThomsonA, McWhirJ. 2003. In vitro osteogenic differentiation of human ES cells. Cloning Stem Cells, 5:149–155.
43.
Zur NiedenNI, KempkaG, RancourtDE, AhrHJ. 2005. Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: effect of cofactors on differentiating lineages. BMC Dev Biol, 5:1.
44.
GadueP, HuberTL, PaddisonPJ, KellerGM. 2006. Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells. Proc Natl Acad Sci U S A, 103:16806–16811.
45.
KärnerE, BackesjoCM, CedervallJ, SugarsRV, Ahrlund-RichterL, WendelM. 2009. Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro. Biochim Biophys Acta, 1790:110–118.
46.
KuznetsovSA, MankaniMH, RobeyPG. 2011. In vivo formation of bone and haematopoietic territories by transplanted human bone marrow stromal cells generated in medium with and without osteogenic supplements. J Tissue Eng Regen Med.[Epub ahead of print]10.1002/term.515.
47.
AldahmashA, ZaherW, Al-NbaheenM, KassemM. 2012. Human stromal (mesenchymal) stem cells: basic biology and current clinical use for tissue regeneration. Ann Saudi Med, 32:68–77.
48.
KennedyM, D'SouzaSL, Lynch-KattmanM, SchwantzS, KellerG. 2007. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood, 109:2679–2687.
WheatleySC, IsackeCM, CrossleyPH. 1993. Restricted expression of the hyaluronan receptor, CD44, during postimplantation mouse embryogenesis suggests key roles in tissue formation and patterning. Development, 119:295–306.
51.
PignoloRJ, KassemM. 2011. Circulating osteogenic cells: implications for injury, repair, and regeneration. J Bone Miner Res, 26:1685–1693.
52.
BrownSE, TongW, KrebsbachPH. 2009. The derivation of mesenchymal stem cells from human embryonic stem cells. Cells Tissues Organs, 189:256–260.
53.
QiuW, AndersenTE, BollerslevJ, MandrupS, AbdallahBM, KassemM. 2007. Patients with high bone mass phenotype exhibit enhanced osteoblast differentiation and inhibition of adipogenesis of human mesenchymal stem cells. J Bone Miner Res, 22:1720–1731.
54.
HarknessL, TaipaleenmakiH, MahmoodA, FrandsenU, SaamanenAM, KassemM, AbdallahBM. 2009. Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker. Stem Cell Rev, 5:353–368.
55.
JukesJM, BothSK, LeusinkA, SterkLM, van BlitterswijkCA, deBJ. 2008. Endochondral bone tissue engineering using embryonic stem cells. Proc Natl Acad Sci U S A, 105:6840–6845.
56.
ProkhorovaTA, HarknessLM, FrandsenU, DitzelN, BurnsJS, SchroederHD, KassemM. 2008. Teratoma Formation by Human Embryonic Stem Cells is site-dependent and enhanced by the presence of Matrigel. Stem Cells Dev, 18:47–54.
57.
BlumB, BenvenistyN. 2007. Clonal analysis of human embryonic stem cell differentiation into teratomas. Stem Cells, 25:1924–1930.
58.
LeorJ, GerechtS, CohenS, MillerL, HolbovaR, ZiskindA, ShacharM, FeinbergMS, GuettaE, Itskovitz-EldorJ. 2007. Human embryonic stem cell transplantation to repair the infarcted myocardium. Heart, 93:1278–1284.
59.
BrederlauA, CorreiaAS, AnisimovSV, ElmiM, PaulG, RoybonL, MorizaneA, BergquistF, RiebeIet al.2006. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells, 24:1433–1440.
60.
BourneS, PolakJM, HughesSP, ButteryLD. 2004. Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting. Tissue Eng, 10:796–806.
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