Injury mechanism dictates contribution of bone marrow-derived cells to murine hepatic vascular regeneration.

Stem and progenitor cells derived from adult marrow have been shown to regenerate vascular cells in response to injury. However, it is unclear whether the type of injury dictates the contribution of such cells to neovascularization and which subpopulations of cells contribute to vascular regeneration. To address these questions, we determined the extent that hematopoietic stem cells (HSC) contributed to blood vessel formation in response to two types of liver injury, partial hepatectomy (PH) and toxin-induced injury. Lac-Z-labeled HSC were engrafted into lethally irradiated, genetically matched recipients. After 14 d, we identified transplanted cells engrafted within the vascular endothelium of toxin-damaged liver, but not in the vasculature of liver regenerated in response to PH. Engraftment of HSC-derived cells occurred in a gradient fashion with the highest activity in the severely injured areas. Although HSC-derived cells contributed to both microvessels and large vessels, the large caliber vessels trended toward higher engraftment levels. Thus, the contribution of marrow-derived cells to hepatic neovascularization is dependent upon the type of injury sustained. Furthermore, following toxin-induced liver injury, engraftment rates trended higher in large vessels compared with capillaries, suggesting that remodeling of existing vessels is a predominant mechanism of repair, relative to the formation of new microvasculature.

Differential mRNA processing in hematopoietic stem cells.

Hematopoietic stem cells (HSCs) maintain tissue homeostasis by rapidly responding to environmental changes. Although this function is well understood, the molecular mechanisms governing this characteristic are largely unknown. We used a sequenced-based strategy to explore the role of both transcriptional and post-transcriptional regulation in HSC biology. We characterized the gene expression differences between HSCs, both quiescent and proliferating, and their differentiated progeny. This analysis revealed a large fraction of sequence tags aligned to intronic sequences, which we showed were derived from unspliced transcripts. A comparison of the biological properties of the observed spliced versus unspliced transcripts in HSCs showed that the unspliced transcripts were enriched in genes involved in DNA binding and RNA processing. In addition, levels of unspliced message decreased in a transcript-specific fashion after HSC activation in vivo. This change in unspliced transcript level coordinated with increases in gene expression of splicing machinery components. Combined, these results suggest that post-transcriptional regulation is important in HSC activation in vivo.

Plasticity and tissue regenerative potential of bone marrow-derived cells.

Diverse in vivo studies have suggested that adult stem cells might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. This idea of stem cell "plasticity" has led investigators to hypothesize that, similar to embryonic stem cells, adult stem cells might have unlimited tissue regenerative potential in vivo, and therefore, broad and novel therapeutic applications. Since the beginning of these observations, our group has critically examined these exciting possibilities for mouse bone marrow-derived cells by taking advantage of well-characterized models of tissue regeneration, Cre/lox technology, and novel stem cell isolation protocols. Our experimental evidence does not support plasticity of hematopoietic stem cells as a frequent physiological event, but rather indicates that cell fusion could account for reported cases of hematopoietic stem cell plasticity or "transdifferentiation" in vivo. Our studies highlight the need for meticulous technical controls during the isolation, transplantation, tracking, and analysis of bone marrow-derived cells during in vivo studies on plasticity. Further studies will be necessary to better define experimental conditions and criteria to unequivocally prove or reject plasticity in vivo. In this review, we focus on results from several studies from our laboratory, and discuss their conclusions and implications.

Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts.

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.

Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates.

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.

Altered phenotype and reduced function of muscle-derived hematopoietic stem cells.

OBJECTIVE: Skeletal muscle-derived cells have the potential to repopulate the major peripheral blood lineages of lethally irradiated mice and thus behave like hematopoietic stem cells (HSC). We have recently shown that muscle cells with HSC activity (ms-HSC) express CD45 and Sca-1, suggesting a hematopoietic origin. Here we sought to clarify contradictions in the literature regarding the phenotype of ms-HSC and precisely define the hematopoietic origin of these cells. METHODS: Skeletal muscle-derived cells fractionated based on the expression of CD45 and c-kit and efflux of Hoechst 33342 and were examined for HSC activity in vivo. WBM HSC expressing beta-galactosidase were transplanted into lethally irradiated recipients, whose ms-HSC compartment was later analyzed for beta-galactosidase activity to determine if ms-HSC were derived from WBM HSC. RESULTS: Muscle-derived HSC fall exclusively in the c-kit(dim)CD45(pos) compartment of the muscle side population (msSP). Furthermore, the CD45(pos) msSP compartment of skeletal muscle is derived from WBM HSC. CD45(pos)c-kit(dim) msSP are about 22-fold less potent in HSC activity than WBM HSC cells in competitive repopulation assays and express low levels of c-kit relative to WBM HSC. CONCLUSIONS: In our transplantation experiments, WBM HSC gave rise to ms-HSC, suggesting that WBM HSC and ms-HSC likely represent the same stem cell population in distinct environments. However, these two related populations are both functionally distinct in their ability to repopulate the peripheral blood of irradiated mice and phenotypically distinct.

Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells.

BACKGROUND AND OBJECTIVES: The aim of this study was to determine whether Hoechst effluxing side population cells isolated from murine liver represent hepatic stem cells, and to examine whether hepatic side population cells arise from bone marrow side population cells. DESIGN AND METHODS: Side population cells were isolated from murine liver by flow cytometry after Hoechst staining and injected directly into murine livers of animals pre-treated with the hepatotoxin 3,5 diethoxy carbonyl-1, 4-dihydrocollidine (DDC). Y-chromosome in situ hybridization was used to track donor cells in the livers. In addition, bone marrow side population cells were stably engrafted into the hematopoietic system of sublethally irradiated recipients and CD45 alleleic staining and Y-chromosome in situ hybridization were used to track side population cell progeny in the liver. RESULTS: In vitro, CD45pos and CD45neg hepatic SP cells gave rise to hematopoietic colonies and mixed colonies of hematopoietic and hepatic differentiation. After orthotopic liver cell transplantation, donor hepatic side population cells contributed to the regeneration of mature liver parenchyma and bile duct epithelium. After transplantation of bone marrow side population cells, both CD45pos and CD45neg hepatic side population cells were partially derived from donor stem cells and could be recruited to repair liver damage after treatment with DDC. INTERPRETATION AND CONCLUSIONS: These findings introduce hepatic side population cells as a facultative liver-regenerating population, reveal interchangeability of tissue stem cells at the level of the side population, and suggest that bone marrow-derived side population cells might be exploited for the repair of diseased or damaged liver.

Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during 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.

Stem cells: a minireview.

The identification of adult-derived stem cells which maintain plasticity throughout the course of a lifetime, has transformed the field of stem cell biology. Bone marrow derived hematopoietic stem cells (HSC) are the most well-characterized population of these multipotential cells. First identified for their ability to reconstitute blood lineages and rescue lethally irradiated hosts, these cells have also been shown to differentiate and integrate into skeletal muscle, cardiac myocytes, vascular endothelium, liver, and brain tissue. Various populations of HSC are being studied, exploiting cell surface marker expression, such as Sca-1, c-kit, CD34, and lin; as well as the abilityto efflux the vital dye Hoecsht 33342. Detection of engrafted donor derived cells into various tissue types in vivo is a laborious process and may involve detection of beta-galactosidase via colorimetric reaction or antibody labeling or green fluorescent protein (GFP) via fluorescence microscopy, as well as in situ hybridization to detect the Y-chromosome. Using these techniques, the search has begun for tissue specific stem cells capable of host tissue regeneration, self renewal, and transdifferentiation. Caution is urged when interpreting these types of experiments because although they are stimulating, limitations of the technologies may provide misleading results.

Muscle-derived hematopoietic stem cells are hematopoietic in origin.

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 C57BL/6-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 C57BL/6-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.