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Fragestellung: Eine vollständige funktionale Regeneration skelettaler Defekte durch multipotente Stamm- und Vorläuferzellen erfordert, dass differenzierende Zellen die spezifische Mechanokompetenz des jeweiligen Zielgewebes erlangen. Am Beispiel der Knorpelneogenese fragten wir hier, ob die [zum vollständigen Text gelangen Sie über die oben angegebene URL]

Knochen und Knorpel geben dem Körper Halt. Doch wie entstehen die beiden wichtigen Stützstrukturen? Heidelberger Forscher:innen haben die molekularen Weichen für die Entwicklung in Richtung „eher hart“ oder „eher weich“ erkannt. Ihre Erkenntnisse eröffnen neue Chancen für die regenerative Medizin, etwa für eine effiziente Behandlung von Knorpelschäden mit Biomaterialien., Ruperto Carola, Nr. 20 (2022): Weich & Hart

Additional file 1. Supplementary Figures S1-S4 and Supplementary Table S1.

Mesodermal differentiation of induced pluripotent stem cells (iPSCs) in vitro and subsequent specification into mesodermal derivatives like chondrocytes is currently afflicted with a substantial cell loss that severely limits tissue yield. More knowledge on the key players regulating mesodermal differentiation of iPSCs is currently needed to drive all cells into the desired lineage and to overcome the current need for intermediate cell selection steps to remove misdifferentiated cells. Using two independent human iPSC lines, we here report that a short initial WNT/β-catenin pulse induced by the small molecule CHIR99021 (24 h) enhanced expression of mesodermal markers (PDGFRα, HAND1, KDR, and GATA4), supported the exit from pluripotency (decreased OCT4, SOX2, and LIN28A) and inhibited ectodermal misdifferentiation (reduced PAX6, TUBB3, and NES). Importantly, the initial CHIR pulse increased cell proliferation until day 14 (five-fold), adjusted expression of adhesion-related genes (CDH3 up, CDH6 down) and increased extracellular matrix (ECM)-related gene expression (COL6, COL1, COL3, COL5, DCN, NPNT, LUM, MGP, MATN2, and VTN), thus yielding more matrix-interacting progenitors with a high aggregation capability. Enhanced contribution to chondrogenic pellet formation increased the cell yield after eight weeks 200-fold compared to controls. The collagen type II and proteoglycan-positive area was enlarged in the CHIR group, indicating an increased number of cartilage-forming cells. Conclusively, short initial WNT activation improved mesoderm commitment and our data demonstrated for the first time to our knowledge that, acting via stimulation of cell proliferation, ECM expression and cell aggregation, WNT pulsing is a key step to make cell selection steps before chondrogenesis obsolete. This advanced understanding of the WNT/β-catenin function is a major step toward robust and efficient generation of high-quality mesodermal progenitors from human iPSCs and toward rescuing low tissue yield during subsequent in vitro chondrogenesis, which is highly desired for clinical cartilage regeneration, disease modeling and drug screening.

Additional file 1: Supplementary Table S1. List of qRT-PCR primers used in this study. Supplementary Table S2. List of cloning primers used in this study. Fig. S1. miR-218 transfection efficiency in SaOS-2 cells. SaOS-2 cells were transiently transfected with miR-218 (grey), or with non-targeting control (NC, black) mimic, and transfection efficiency was controlled by qRT-PCR. Mean values ± SD are shown, n = 2; ***p ≤ 0.001, two-tailed t-test. Fig. S2. miR-218 transfection efficiency in human MSC. MSC cells (N = 3 donors) were transiently transfected with miR-218 (grey), or with non-targeting control (NC, black) mimic before cells were subjected to chondrogenic differentiation for 28 days, and transfection efficiency was controlled by qRT-PCR at indicated time points. Mean values ± SD are shown; *p ≤ 0.05, NC vs miR-218; expression kinetic curves were compared using two-tailed paired t-test. Fig. S3. Gain of function of miR-218 at the start of chondrogenic differentiation does not additionally boost MSC chondrogenesis. MSC (N = 4 - 6) were transfected with miR-218 (grey), or with a control non-targeting miR (NC, black) mimics at day 0, and subjected to chondrogenic differentiation for 28 days. a, Safranin O staining of 3D pellet cultures at day 28 chondrogenesis. Scale bar, 500 μm. b, Total GAG and DNA content of 3D pellet cultures was assessed by DMMB and PicoGreen assays at day 28 of chondrogenesis; Mean values ± SD are shown; N = 6 donors.