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The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues

Maria G. Minasi1,2,*, Mara Riminucci3,*, Luciana De Angelis2,*, Ugo Borello1, Barbara Berarducci1, Anna Innocenzi1, Arianna Caprioli4, Dario Sirabella2, Marta Baiocchi2,5, Ruggero De Maria5, Renata Boratto6, Thierry Jaffredo4, Vania Broccoli1, Paolo Bianco7,{dagger} and Giulio Cossu1,2,{dagger}

1 Stem Cell Research Institute, Dibit, H. S. Raffaele, Via Olgettina 58, 20132 Milano, Italy
2 Dipartimento di Istologia ed Embriologia Medica, Universita’ di Roma ‘La Sapienza’ Via Scarpa 14, 00161 Roma, Italy
3 Dipartimento di Medicina Sperimentale, Università de L’Aquila, 67100 L’Aquila, Italy
4 Institut d’Embryologie Cellulaire et Moleculaire du CNRS et du College de France; 49 bis, Avenue de la Belle Gabrielle, Nogent sur Marne, France
5 Laboratorio di Ematologia Oncologia, Istituto Superiore di Sanità, Viale Elena 297, 00161 Roma, Italy
6 Dipartimento di Medicina Sperimentale, Università di Pavia, Via Forlanini 8, 27100 Pavia, Italy
7 Dipartimento di Medicina Sperimentale e Patologia, Universita’ degli Studi di Roma ‘La Sapienza’, Viale Regina Elena 324, 00161 Roma, Italy
* These authors contributed equally to this work



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  		Fig. 1. Immunoperoxidase staining with QCPN antibody of chick-quail chimeras sacrificed at E19-E20. Regular arrays of quail nuclei (dark brown, arrowheads) are detected in the vessel wall of blood vessels of large (A), intermediate (B) and small (C) sizes. Donor nuclei are detected in pre-capillary arterioles, adjacent the base of a feather bud (arrowhead) and inside the bud (D), as well as circulating cells in the vessel lumen (E). Quail nuclei are detected in cutaneous smooth muscle in quail-chick chimeras, as seen in longitudinal (SM-L in F) and transverse (SM-T in G) sections. (H) Hematoxylin and Eosin staining of a similar section showing morphology of bundles of smooth muscle associated with feather buds (F), immunostained for {alpha}-smooth actin (J).

 


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  		Fig. 2. Immunoperoxidase staining with QCPN antibody of chick-quail chimeras sacrificed at E19-20. (A,B) Detection of quail nuclei in the osteogenic perichondrium at leading edge of periosteal ossification. (A) Alkaline phosphatase staining, ALP; (B) QCPN immunostaining. Note the marked reactivity for ALP of multiple cell layers in the richly vascular osteogenic perichondrium, but not in the adjacent fibroblastic layer. QCPN immunoreactive cells reside within the ALP-positive layer and cluster around thin-walled blood vessels (bv). Arrows in B indicate quail nuclei. (C,D) More advanced stage of bone deposition, marked by a recognizable bony collar (arrows). C, ALP staining; D, QCPN immunostaining. Note the presence of quail nuclei within the boundaries of the ALP-positive osteogenic layer of the perichondrium/periosteum. (E,F) Quail chondrocytes are observed in superficial (sub-perichondral) regions of hyaline cartilage (E, QCPN immunostaining; F, QCPN immunostaining/Alcian Blue), and in chondrocytes surrounding the marrow cavity and vascular canals (G). Quail nuclei also occur frequently in hematopoietic cells (hc) in the bone marrow (H,I). Adjacent HE stained (H) and QCPN immunostained (I) sections depicting the hematopoietic tissue in the marrow cavity of a long bone. (J) QCPN-immunoreactive osteocyte (arrowhead) residing in a typical osteocytic lacuna in well-formed bone.

 


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  		Fig. 3. Immunoperoxidase staining with QCPN antibody of chick-quail chimeras sacrificed at E19-E20. Quail nuclei (dark brown) are detected within and around the wall of intramuscular blood vessels (arrow in A) and also among and sometimes adjacent to developing muscle fibers. Quail nuclei are also seen inside cross-striated muscle fibers (B,C). (D,E) Double immunofluorescence staining with QCPN antibody reveals quail nuclei (red) in skeletal (D) and cardiac (E) muscle cells that express sarcomeric myosin (green). Quail nuclei appear red in the interstitium and orange when inside myosin-positive cells. Scale bar: 25 µm.

 


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  		Fig. 4. X-gal staining of mouse-chick chimeras sacrificed at E19. Mouse nuclei that have activated the MLC1/3F-nlacZ reporter gene nuclei (blue in A) are detected inside myosin-positive muscle fibers (A,B). Higher magnification in the inset in A shows lacZ-positive nuclei inside fibers. Donor lacZ-positive cells are also detected in myosin-positive cardiocytes (C,D) of mouse-chick chimeras sacrificed at E19. Mouse donor cells that were sorted for PECAM and labeled with DiI were similarly detected in the host chick heart (E), blood vessels (F) and cartilage (G): E, merged DiI (red) and myosin heavy chain (green) fluorescence images; F, merged DiI (red) and desmin (green) fluorescence images; and G, merged DiI (red) and DAPI (blue) fluorescence images.

 


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  		Fig. 5. (A) Morphology of the embryonic dorsal aorta isolated from E9.5 mouse embryos after pancreatin digestion. Note the absence of remnants of adjacent embryonic structures. (B) RT-PCR revealed that endothelial and hematopoietic markers (VE-Cad, Flk1 and CD34), but not myogenic markers (Myf5 and MyoD), were expressed in dissected aorta (A). Total embryo extract was used as a positive control (+). Negative control (no RNA) is shown in the first lane (–). (C) Phase contrast morphology of a clone from embryonic aorta growing on an STO feeder layer. (D) Phase contrast morphology of one typical clone (A4) from embryonic aorta after five passages in vitro. (E) RT-PCR of the messages expressed by several cell lines (A4, A6, A14 and B13) from the dorsal aorta after 5 passages in vitro. Note expression of hemo-angioblastic but not of tissue specific markers such as Myf5 or Nkx2.5.

 


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  		Fig. 6. In vitro differentiation of the aorta derived cell line A4 into different cell types. (A) Smooth muscle cells (SMA positive, arrowheads). (B) Osteoblasts (ALP positive, arrowheads) are detected after treatment with 1 ng/ml BMP2; (C) Adipocytes are detected after treatment with 10 ng/ml of dexamethasone. (D) Skeletal myotubes are detected after co-culture of GFP-labeled A4 cells with C2C12 myoblasts. GFP-positive cells appear green, myocytes and myotubes expressing myosin heavy chains appear red, and cells expressing both appear yellow in the merged image (arrowhead). A mononucleated, differerentiated, GFP-positive myocyte is shown in the inset in D. (E) Cardiocytes are detected after co-culture of GFP-labeled A4 cell with rat neonatal cardiocytes. GFP-positive cells appear green, myocardiocytes expressing cardiac specific troponin 1 appear red and cells expressing both appear yellow in the merged image (arrowhead).

 


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  		Fig. 7. (A) Histogram showing percentages of GFP-labeled, A4 cells (blue bars) or of GFP-labeled A4 cells co-cultured with wild-type adult bone marrow in a Dexter-type culture (red bars) after FACS sorting for expression of Ter119, CD45, Mac3, CD11b and Gr1. (B) A multinucleated (arrowhead), and another TRAP-positive cell are shown from a culture treated with 1,25 (OH)2 vitamin D3. (C) RT-PCR for the expression of the calcitonin receptor in control cells (c) and cells treated (t) as described above. (D-G) Morphology of GFP-positive cells, stained with Giemsa, revealing predominantly blast (D,F), macrophage (E) and immature myeloid (G) morphologies.

 


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  		Fig. 8. Chick-mouse chimeras (sacrificed at E14) that had been transplanted with aorta-derived clonal cell line A4, at passage 25. (A) Whole-mount fluorescence of the transplant site, showing a cluster of GFP+ donor cells. (B) Section through a large vessel showing donor cells integrated into a large vessel wall (arrowhead) stained for smooth {alpha}-actin. (C) A cluster of donor cells outside a small vessel, with one cell (arrowhead) double expressing GFP and SMA (yellow in the merged image). Inset: GFP circulating cells inside a small vessel (arrowhead). (D) Donor cells accumulate in the dermis but not in the epidermis (arrowhead). Merged image: GFP (green), Hoechst (blue) and myosin heavy chains (red) of an adjacent muscle primordium containing no donor cells. (E) Section through the myocardium showing several donor cells (yellow) in the myocardium (arrowhead) and in the sub-epicardium. (F) Section through the intestine showing donor cells in the smooth muscle layer (arrowhead) stained for SMA. (G) Section through axial tissues showing donor cells in the cartilage adjacent to the perichondrium (arrowhead), in the perichondrium (asterisk) and in the blood vessel (arrow), but not in the intervening muscle fibers. (H) Section through a muscle primordium, showing donor cells (green) intermixed with myosin heavy chain-positive muscle fibers (red). Arrowhead indicates double-labeled fibers.

 




© The Company of Biologists Ltd 2002