Constitutively active (ca) caALK5 and caALK1 receptors do not induce BMSC chondrogenesis as efficiently as TGFβ does.

<p>Human fetal BMSCs (donor F1) transduced with adenoviral caALK5, caALK1 or LacZ as control were pellet-cultured in chondrogenic medium for 7 days. Only LacZ-transduced pellets were exposed to TGFβ to compare if either caALK5 or caALK1 induced chondrogenesis as efficiently as induced by TGFβ. Chondrogenesis was evaluated by cartilage-specific gene expression analysis of <i>ACAN</i> <b>(A)</b> and <i>COL2A1</i> <b>(B)</b> and by staining proteoglycans with Safranin O/Fast Green <b>(C; upper panel)</b> and collagen type II with immunohistochemistry <b>(C; lower panel)</b>. Representative images of consecutive pellet sections per condition are shown and the scale bar represents 500 μm. Gene expression was normalized to reference gene <i>RPS27a</i> and data are expressed as % relative to normalized gene levels in LacZ-transduced pellets stimulated with TGFβ. Bars represent mean ± SD from quadruplet pellets of 1 representative experiment (out of 3), ***p<0.001.</p

Gene expression levels of ALK5 <i>(TGFBR1)</i> and ALK1 <i>(ACVRL1)</i> are not associated with the chondrogenic capacity of human BMSCs.

<p>To induce chondrogenesis, BMSCs (passage 2–4) from adult donors A1 –A5 and fetal donor F1 were pellet-cultured in chondrogenic medium and stimulated with TGFβ. Cartilage-specific gene expression of aggrecan <i>(ACAN)</i> <b>(A)</b> and collagen type IIα1 (<i>COL2A1)</i> <b>(B)</b> was determined in BMSCs cultured for 0, 1, 4, 7 and 21 days using RT-qPCR. Cartilage-specific protein depositions of proteoglycans <b>(C; upper panel)</b> and collagen type II <b>(C; lower panel)</b> were stained in sections of 21 days-cultured pellets. To stain proteoglycans, sections were stained with Safranin O (red/orange) and Fast Green (blue) was used as counterstaining. Collagen type II was stained immunohistochemically (pink) and hematoxylin (purple) was used as counterstaining. Representative images of consecutive pellet sections per donor are shown and the scale bar represents 500 μm. <i>TGFBR1</i> <b>(D)</b> and <i>ACVRL1</i> <b>(E)</b> expression levels were measured in BMSC monolayer (day 0) and pellets cultured for 1, 4 and 7 days. Gene expression was normalized to reference gene <i>RPS27a</i>. Data points represent mean ± SD of triplicate BMSC pellets per donor per time point.</p

List of primers used for RT-qPCR.

<p>List of primers used for RT-qPCR.</p

BMSCs overexpressing either constitutively active (ca) caALK5 or caALK1 express enhanced levels of phosphorylated SMAD2/3 and SMAD1/5/8 proteins, respectively.

<p>Human fetal BMSCs (donor F1) were transduced with adenoviral caALK5, caALK1 or LacZ as control and pellet-cultured in chondrogenic medium for 1 day. Transduction efficiency of caALK5 and caALK1 was evaluated after gene expression analysis of <i>TGFBR1</i> <b>(A)</b> and <i>ACVRL1</i> <b>(B)</b>. Gene expression was normalized to reference gene <i>RPS27a</i> and data are expressed as fold change relative to normalized gene levels in LacZ-transduced BMSCs. Bars represent mean ± SD from triplicate pellets of 1 representative experiment (out of 3), **p<0.01; ***p<0.001. Constitutively active receptor signaling of ALK5 and ALK1 was evaluated by Western blot analyses of phosphorylated SMAD (pSMAD) proteins; pSMAD2/3 <b>(C)</b> and pSMAD1/5/8 <b>(D)</b> using GAPDH as loading control.</p

TGFβ increases <i>TGFBR1</i> (ALK5) and decreases <i>ACVRL1</i> (ALK1) in human BMSC pellets.

<p>TGFβ increases <i>TGFBR1</i> (ALK5) and decreases <i>ACVRL1</i> (ALK1) in human BMSC pellets.</p

ALK5 and ALK1 are expressed in human bone marrow-derived mesenchymal stem cells (BMSCs).

<p>In human BMSCs, obtained from four adult donors (A1 –A4) and one fetal donor (F1), gene expression levels of ALK5 <i>(= TGFBR1)</i> <b>(A)</b> and ALK1 <i>(= ACVRL1)</i> <b>(B)</b> were analyzed using RT-qPCR. Gene expression was normalized to reference gene <i>RPS27a</i>. Each data point represents one measurement.</p