No abstract

We have discovered that cells derived from the skeletal muscle of adult mice contain a remarkable capacity for hematopoietic differentiation. Cells prepared from muscle by enzymatic digestion and 5-day in vitro culture were harvested, and 18 ؋ 10 3 cells were introduced into each of six lethally irradiated recipients together with 200 ؋ 10 3 distinguishable whole bone marrow cells. After 6 or 12 weeks, all recipients showed high-level engraftment of muscle-derived cells representing all major adult blood lineages. The mean total contribution of muscle cell progeny to peripheral blood was 56 ؎ 20% (SD), indicating that the cultured muscle cells generated approximately 10-to 14-fold more hematopoietic activity than whole bone marrow. When bone marrow from one mouse was harvested and transplanted into secondary recipients, all recipients showed high-level multilineage engraftment (mean 40%), establishing the extremely primitive nature of these stem cells. We also show that muscle contains a population of cells with several characteristics of bone marrow-derived hematopoietic stem cells, including high efflux of the fluorescent dye Hoechst 33342 and expression of the stem cell antigens Sca-1 and c-Kit, although the cells lack the hematopoietic marker CD45. We propose that this population accounts for the hematopoietic activity generated by cultured skeletal muscle. These putative stem cells may be identical to muscle satellite cells, some of which lack myogenic regulators and could be expected to respond to hematopoietic signals. Regenerative stem cells can be found in many adult tissues (1-6). Although possessing substantial capacity to proliferate and differentiate, such cells are thought to be committed to differentiate exclusively into the tissues in which they reside. However, recent reports have suggested that some ostensibly tissue-specific progenitors may have differentiation potential outside of their tissue of origin. Ferrari et al. (7) found that lacZ-marked cells derived from bone marrow of donor mice could be incorporated into regenerating skeletal muscle of recipients. After bone marrow transplantation, donor-derived cells have also been found in multiple nonhematopoietic tissues, including liver (8), vascular endothelial cells (9), astroglia in the brain (10), skeletal muscle (7, 11), and bone (12). Although bone marrow contains many cell types that could account for this variety of activities, it is possible that hematopoietic stem cells (HSC) are directly or indirectly involved.Moreover, stem cells derived from nonhematopoietic tissue have been found to differentiate into hematopoietic cells. Bjornson et al. (13) showed that clonal populations of neural stem cells could repopulate the hematopoietic system after bone marrow transplantation. Together, these studies suggest that stem cells derived from adult tissues may retain a previously unrecognized degree of plasticity in their commitment and that their differentiation may be influenced more by environment than by lineage.This possibility led us t...

It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6-to 8-week-old C57BL͞6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL͞6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.S tem cells are defined by their ability to self renew and differentiate into the cell types of their derivative tissue. Traditionally, it has been assumed that a stem cell derived from adult tissues can give rise only to progeny specific to that tissue type. However, this dogma has been challenged recently by a series of studies that suggest that adult tissue-derived stem cells may have the potential to differentiate into disparate cell types. For example, purified hematopoietic stem cells (HSCs), derived from whole bone marrow (WBM), have been shown to contribute to regenerating skeletal muscle (1), cardiac muscle (2), liver (3), and multiple epithelial tissues (4). In addition, stem cells from other tissues have also been proposed to differentiate outside their tissue of origin (5, 6). Although these studies have provoked new critical thinking about stem cell differentiation capacity, definitive proof of transdifferentiation remains to be established at a clonal level.Two recent studies focused on the ability of muscle-derived cells to repopulate WBM in lethally irradiated mice. Gussoni et al. (1) reported that muscle cells fractionated on the basis of their efflux of Hoechst dye could rescue lethally irradiated recipients. Similarly, Jackson et al. (7) reported that unfractionated mononuclear muscle cells could repopulate all major blood lineages of lethally irradiated mice up to 12 weeks after transplant. In addition, when bone marrow from engrafted animals was transplanted into secondary recipients, their peripheral blood was also repopulated with muscle-derived cells, demonstrating the important property of self renewal (7).Satellite cells are a potent myogenic stem cell population that resides in the muscle and are responsible for postnatal muscle regeneration and growth (8, 9). We proposed that satellite cells accounted for muscle-derived hematopoietic activity via transdifferentiation when introduced...

Recent studies have shown that cells from the bone marrow can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved have remained unknown, and the validity of the observation has been questioned. Here, we use transplantation of single CD45+ hematopoietic stem cells (HSCs) to demonstrate that the entire circulating myogenic activity in bone marrow is derived from HSCs and their hematopoietic progeny. We also show that ongoing muscle regeneration and inflammatory cell infiltration are required for HSC-derived contribution, which does not occur through a myogenic stem cell intermediate. Using a lineage tracing strategy, we show that myofibers are derived from mature myeloid cells in response to injury. Our results indicate that circulating myeloid cells, in response to inflammatory cues, migrate to regenerating skeletal muscle and stochastically incorporate into mature myofibers.

Reports have suggested that adult mouse bone marrow cells (BMCs) are capable of transdifferentiating into cells with neural characteristics in the central nervous system (CNS) (1, 2). Because side-population (SP) cells within whole bone marrow are hematopoietic stem cells that can reconstitute the BMC population and are capable of differentiating into other types of cells such as cardiac myocytes and endothelial cells (3-5), wecells. Their close association with blood vessels and the lack of morphological features of neural cells suggested that they were hematopoietic. The brains from Rosa26 control mice had robust P-Gal staining in neural as well as blood cells.Because injury increases SP transdifferentiation in other tissue systems (5), we tested whether neural injury would cause donor cells to transdifferentiate into neural-like cells. Four Table 1. Summary of experimental manipulations and results. ND, not done. Type and number of mice receiving injury to the Brains with Type of cells brain donor-derived transplanted neural cells/brains Contusion injury Stab injury No injury analyzed SP cells 2 4 2 0/8* BMCs ND 7 5 0/12* *50 to 100 coronal sections containing more than 106 neural cells were analyzed per brain.surmised that they too would transdifferentiate into neural cells.To test this, 8 C57B1/6 (B6) mice were treated with a lethal dose of irradiation and transplanted with 2 X 103 SP cells derived from the Rosa26 mouse. The Rosa26 mouse carries the LacZ gene that is constitutively expressed in most cells including neural and SP cells and is an unambiguous marker for donor-derived cells (6). Ten to 12 weeks after transplantation, 80 to 95% of the recipient blood cells were LacZ positive. Four months after transplantation, the CNS of two of the recipient mice were inspected for cells derived from the Rosa26 donor with standard X-gal cytohistochemistry. In coronal sections (50 to 100 sections per brain representing more than 106 cells per brain) taken throughout the full extent of the brain, including the olfactory bulbs and cervical spinal cord, the only P-galactosidase (P-Gal)-positive cells detected were a few cells (<5) that were associated with blood vessels. These P-Gal-positive cells had a globular morphology and no processes that would suggest that they were neural mice with SP transplants underwent cortical stab injury, and two underwent cortical contusion injury (7) 4 months after the transplant. Reconstitution of the hematopoietic system is considered stable and complete around 4 months after BMC or SP transplantation. Therefore, cells capable of transdifferentiation should be in place at that time. One month after cortical stab injury and 4 months after contusion injury, no 3-Gal-positive cells were observed in brains (fig. S1) or cervical spinal cords except a rare few associated with blood vessels. We concluded that adult bone marrow SP cells or cells derived from them were incapable of transdifferentiating into neural cells in this experimental system.Because SP cells will reconstitute the BMC p...

Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration

Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: side population (SP); plateletendothelial cell adhesion molecule-1 (PE-CAM-1); transcription factor Scl/Tal-1 (Tal 1); VEGF receptor-1 (Flt-1); VEGF receptor-2 (Flk-1); angiopoietin-1 (Ang-1); tyrosine kinase with immunoglobulin and epidermal growth factor homology-1 (Tie-1); vascular-endothelial cadherin (VE-cadherin); smooth muscle α actin (SM-α-actin); fluorescein-di-β-Dgalactopyranoside (FDG); tibialis anterior (TA).

Adult stem cell research has lately been plagued by controversy regarding the possibility that some adult stem cells can engraft into nonautochthonous tissues. While most reports have observed some level of engraftment, the prevalence has varied in some cases by two orders of magnitude, suggesting that major technical variations may underlie these differences, possibly outweighing the biological basis of the observations. Here we describe bright green autofluorescence in a specific subset of skeletal muscle fibers that strongly resembles emission from green fluorescent protein (GFP). Moreover, we show that oxidative muscle fibers exhibit this autofluorescence, likely due to flavin, associated with NADH dehydrogenase. Finally, we demonstrate that confocal microscopy, in conjunction with spectral scanning, can be used to distinguish between GFP and autofluorescence. We suggest this autofluorescence artifact may account for some of the discrepancies in this field, particularly those describing skeletal muscle engraftment.