Ono et al. 10.1073/pnas.0704472104. |
Fig. 6. Flow cytometric analysis of myoSP and myoMP. (A) Expression patterns of endothelial cell surface markers (CD106, vascular endothelial growth factor-1 [VEGFR-1], Factor VIIIra [Factor VIII-related antigen]) in myoSP (red) and myoMP (blue). (B) Expression patterns of mesenchymal stem cell surface markers (CD90, CD44, CD73, CD105, STRO-1) in myoSP (red) and myoMP (blue). Mouse FITC-labeled IgG1, FITC-labeled IgG2a, APC-labeled IgG1, and PE-labeled IgG1 (all from BD Biosciences) were used as isotypic controls for staining of total myometrial cells (black).
Fig. 7. Coexpression of human nuclear antigen (HNA) and human Vm in myoSP-derived tissues in the E2-treated uteri of NOG mice. NOG mice were ovariectomized and xenotransplanted with myoSP into their uteri, s.c. implanted with an E2 pellet, and hysterectomized 10 weeks after transplantation. The excised uteri were subjected to immunofluorescence staining and confocal microscopy using antibodies against HNA and Vm, followed by TOTO3 staining. Arrowheads indicate coexpression of Vm (red) and HNA (green) shown for several selected cells in side views of confocal stack.
Fig. 8. Confocal three-dimensional reconstruction images of the pregnant NOG mouse uterus transplanted with myoSP. Serial sections were stained with antibodies against aSMA, Vm, and OTR as indicated. Arrowheads indicate the colocalization (yellow) of aSMA and Vm (white arrowheads) or OTR and Vm (blue arrowheads).
Fig. 9. Induction of several bone-differentiation markers in cultures of myoSP, but neither myoMP nor total myometrial cells, in the presence of osteocyte-inducing media. The indicated cells were treated with control media (-) or osteocyte-inducing media (+) for 3 weeks, harvested, and subjected to RT-PCR analysis for mRNA expression of osteocalcin (OC), core binding factor a1 (CBFA-1), alkaline phosphatase (ALP), parathyroid hormone-receptor 1 (PTHR-1), and GAPDH.
Fig. 10. No reconstitution of human-originated tissues and myofibers in myoSP-transplanted tibialis anterior (TA) muscles in NOG mice. TA muscles were chemically injured by a single 10-ml injection of notexin (Latoxan, 10 mg/ml) and thereafter transplanted with myoSP (5 ´ 104 cells). After 4 weeks, they were excised, snap-frozen, and subjected to immunofluorescence staining and confocal microscopy using antibodies against laminin (Sigma) or Vm followed by TOTO3 staining. Note that Vm-positive cells were absent in the myoSP-transplanted TA muscles. Similarly, myoMP generated neither Vm-positive tissues nor myofibers (data not shown).
SI Methods
FACS Analysis.
Myometrial cells were sorted by a FACS Vantage SE flow cytometer (BD Biosciences) and analyzed with Cell-Quest software (BD Biosciences). The FACS Vantage SE flow cytometer is equipped with an argon laser (488-nm excitation), a HeNe laser (633-nm excitation), and a multiline UV laser (334- to 364-nm-excitation). Hoechst33342 was excited at 350 nm, and fluorescence emission was measured at both 424/44 nm (Hoechst blue) and above 670 nm (Hoechst red). A 610-nm short-pass dichroic mirror was used to separate the emission wavelengths. Both Hoechst red and Hoechst blue fluorescence are shown on a linear scale. After collecting 5 ´ 104 events, the SP and MP populations were defined as reported (1, 2).Cell Culture.
Both myoSP and myoMP were cultured in MSC growth medium (MSCGM; Cambrex Bio Science) under normoxic (20% O2) or hypoxic (2% O2) conditions for 4 weeks. Cell proliferation activities were measured by using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's instructions.For induction of differentiation, myoSP and myoMP were plated at a density of »5 ´ 103 cells per well in 96-well dishes with MSCGM and grown in a hypoxic environment until the cells reached confluence (14-28 days). For osteogenic induction, the cultures were then exported to a normoxic environment, fed with osteocyte differentiation media (Cambrex Bio Science) every 3-4 days for 2-3 weeks. The cells were then harvested for RNA extraction or subjected to alkaline phosphatase staining using the Histofine New Fuchsin Substrate kit (Nichirei). For adipogenic induction, confluent cultures of myoSP and myoMP were exported to a normoxic environment and exposed to three cycles of incubation with adipogenic induction/maintenance media (Cambrex Bio Science). Each cycle consisted of incubation with the supplemented adipogenesis induction media for 3 days, followed by 1- to 3-day incubation with adipogenic maintenance media. After three cycles of incubation, cells were cultured for at most another week in the maintenance media and then harvested for RNA extraction or subjected to Oil red O staining.
1. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) J Exp Med 183:1797-806.
2. Matsuzaki Y, Kinjo K, Mulligan RC, Okano H (2004) Immunity 20:87-93.