Cell Surface Expression of Stem Cell Antigen-1 (Sca-1) Distinguishes Osteo-, Chondro-, and Adipoprogenitors in Fetal Mouse Calvaria

Abstract

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1+ and Sca-1(-) cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1+ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1(-) cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1(-) fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1+ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1(-) cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1+ cell fraction with adipogenic potential from a Sca-1(-) cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.

Osteogenic and Adipogenic Cell Fractions Isolated from Postnatal Mouse Calvaria

“…Consistent with our recent studies on cells from fetal mouse calvaria [Steenhuis et al, 2008], we show here that the Sca-1 + cell fraction from postnatal mouse calvaria has adipogenic differentiation potential, and that the Sca-1 -cell fraction has osteogenic differentiation potential in vitro and osteochondrogenic differentiation potential in vivo. In contrast to fetal cells, postnatal calvarial cells retain some of their differentiation potential after in vitro expansion, which would be important for possible therapeutic use of calvaria-derived progenitor cells.…”

“…Primary cells within the Sca-1 + cell fraction derived from fetal day 18.5 mouse calvaria had the ability to differentiate into adipose, whereas primary cells within the Sca-1 -cell fraction derived from mouse calvaria at fetal day 18.5 had the ability to differentiate into bone and cartilage [Steenhuis et al, 2008]. These data were intriguing given that a multipotent cell line has previously been isolated from rodent calvaria.…”

“…We recently showed that the stem cell marker Sca-1 is expressed in mouse calvaria throughout development [Steenhuis et al, 2008]. Primary cells within the Sca-1 + cell fraction derived from fetal day 18.5 mouse calvaria had the ability to differentiate into adipose, whereas primary cells within the Sca-1 -cell fraction derived from mouse calvaria at fetal day 18.5 had the ability to differentiate into bone and cartilage [Steenhuis et al, 2008].…”

Understanding heterotopic ossification after spinal cord injury

“…Sorted population negative for Sca-1 and CD34 markers in bone marrow could represent osteochondroprogenitors as have been suggested before [124,245] and therefore it can be already prone to osteogenic fate which explains their high osteogenic differentiation rate and expression of Runx2, Osteocalcin and Osterix, similar to BM Sca-1 + progenitors and MC3T3 cell line population.…”

“…After decontamination of the fur with 70% ethanol, hamstring muscles from naïve mice were collected and dissociated into single cell suspensions by successive digestions with collagenase and dispase as previously described [96]. Briefly muscle tissue was cut into 1- Single cell suspensions isolated from muscles and bone marrow were then stained with biotinylated mouse CD45 and Ter119, CD31-APC, anti-Sca1-PECY7, CD34-FITC, streptavidin-PE following standard procedure [72,123,124]. Two µg/mL 7-aminoactinomycin D was added 10 minutes before sorting in order to exclude dead cells.…”