**1. Introduction**

In many traditional stem/progenitor models for different tissues the generally accepted models have posited a primitive stem cell giving rise to more differentiated progenitors and finally terminally differentiated end cells, which may or may not retain the capacity for cell division. Perhaps the most intensively studied stem cell system has been that of the hematopoietic stem cell [1–6]. In general, current dogma has it that the long-term repopulating hematopoietic stem cell is a dormant non-cycling cell characterized by a surface phenotype that is negative for conventional differentiation markers (B220, Gr-1, Mac-1, Lyt-2, L3T4 and Ter119) and positive for c-kit, Sca-1 and CD150. It is felt that this cell can be purified by FACS and that in response to various differentiating stimuli it progressively differentiates into different lineage restricted populations, which in general are actively cycling. A large number of studies have extensively characterized its molecular regulation

and biologic characteristics [6–11]. We began studying both purified stem cells and unseparated whole marrow stimulated to progress through cell cycle with cytokine exposure and demonstrated that there were cycle related and reversible changes over time in long and short term engraftment, progenitor levels, differentiation into megakaryocytes and granulocytes, homing to marrow, capacity to alter phenotype toward lung cells in response to pulmonary derived extracellular vesicles (EVs), overall gene expression, capacity to take up vesicles and circadian characteristics [12–22]. Passegue and colleagues [23] studied lineage negative Sca-1+ c-kit+ and thy+/− stem cells further separated into G0, G1 and S/G2/M fractions as to longterm engraftment into lethally irradiated mice. They found that all engraftment capacity was in the G0 population. This indicated that our observations might be *in vitro* artifacts. However, we noted that no one had adequately studied unseparated marrow as to the cycle status of long-term repopulating stem cells. We essentially reproduced the Passegue data studying purified stem cells, but when we studied unseparated whole marrow we found that over 50% of the long-term engraftable cells were in the S/G2/M fractions. In order to address the potential problem of cellular cross contamination in the FACS experiments we utilized a thymidine suicide technique in which cycling cells are selectively killed by a 30-minute exposure to high specific activity tritiated thymidine. Studies here showed that anywhere between 70 and 100% of the long-term engrafting stem cells were in S phase at the time of the incubation. Further work using *in vivo* BrdU indicated that the dormant purified stem cells (Lin-C-kit+Sc-1+) rapidly progressed through the cell cycle such that up to 85% of them showed the BrdU label by 48 hours of *in vivo* BrdU exposure. These data showed that a large number of HSC in the mouse are actively proliferating and thus always changing phenotype. When lineage positive and lineage negative marrow cells were assessed for engraftment and cycle status, it was found that a large number of marrow stem cells were in both fractions and were cycling [24]. In further work, we have shown that different lineage positive cells are rich in cycling stem cells, but intriguingly when double sorted that lineage positive cells no longer showed HSC characteristics but there was a separate population negative for the particular marker with enriched and cycling HSC. This data has led to our current hypothesis that hematopoietic stem cells exist on a cycle related continuum and that these cells while maintaining critical stem cell markers show cycle related fluctuation in differentiation markers [22]. We feel that we may be defining the calculus of hematopoietic stem cells with time and cycle related phenotype changes being the derivatives and with the final overall picture the integral.

Whether this model of small incremental cellular changes over time applies to other stem cell systems will clearly be the object of much future work.

The stability of cell types and systems is also up for grabs. Early in the study of hematopoietic stem cells Till et al. [25] showed that the first stem cell assay, the colony forming unit spleen, showed marked heterogeneity but the assay was generally reliable. They compared the CFU-s system to radioactive isotopes; the individual decay rates were totally heterogeneous, but the overall half-lives were reproducible and quite exact. These data would indicate the potential importance of evaluating the total population of stem cells alongside the purified variety. The variable and shifting phenotypes of the stem cell with cell cycle transit has to be considered in the context of extracellular vesicle modulation of cell phenotype.

### **1.1 Extracellular vesicles**

Tiny lipid membrane enclosed particles are released from essentially all cell types in the mammalian body [26, 27]. These entities were first found to come

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DNA breaks.

models.

messages.

*Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease*

from red blood cells and platelets and felt to be essential cellular waste products [28, 29]. Subsequent work has characterized and subdivided these entities to size, density and morphology. Eventually two basic types of vesicles were defined by differential ultracentrifugation; exosomes and microvesicles. Exosomes derived from multivesicular bodies were from 30 to 100 nm in diameter while microvesicles derived from membrane blebbing were from 100 to 1000 nm in diameter. Other vesicular entities were also defined including apoptotic bodies. In general, there was much overlap between exosomes and microvesicular bodies and a meeting of investigators decided it might be best to simply term these as extracellular vesicles (EV) and then define their source and the conditions under which they were isolated. Vesicles were eventually isolated from virtually all bodily fluids and cells [26, 27]. Recent focus has been on the capacity of EV to restore injured tissue and treat disease. Initial work showed that vesicles could transfer protein and RNA while modifying the phenotype of cells and reversing disease in animal

Ratajczak et al. showed that embryonic stem cell derived microvesicles could reprogram hematopoietic progenitors by horizontal transfer of mRNA and protein delivery [30]. This was followed by work by Aliotta et al. [31–33] and Valadi et al. [34] showing RNA transfer and phenotype change in different experimental models. Further work has indicated that cellular phenotype change may be mediated by transfer of transcriptional activators possibly miRNA [35]. Vesicles from different sources are different but do contain some features of their originating cells. Vesicles contain protein, mRNA, miRNA, lipids and variably DNA, thus they represent complex bio machines with a tremendous range of potential phenotype altering

Vesicles have been found to have a variety of effects on both normal and diseased or injured tissues. These effects may be negative or positive depending upon the specifics of the experimental models under consideration. In many instances there appears to be a yin/yang nature to vesicle effects. Studies with murine marrow cells have illustrated the complexity of vesicle marrow cell interactions [26, 27]. Early studies showed that ES derived vesicles could improve proliferative status of lin-Sca-1+ marrow stem cells [30] and work in our laboratory has shown that lung derived EVs could induce expression of surfactant A, B, C and D, Clara cell protein and aquaporin in normal murine marrow cells [31–33]. Studies indicated that for a genomic change to occur the vesicle had to enter the target marrow cells [34] and that initially both mRNA and a transcriptional regulator were transferred to target cells but that the transferred mRNA was degraded and long-term expression of surfactants B and C (those tested in these studies) derived from the target cells and represented a stable epigenetic event [35]. The functional effects of vesicles on marrow mRNA expression depended upon the cell cycle status of the target marrow cells and the condition of the originator lung cells, in this case either irradiated or not [21]. The results showed that Lin-Sca-1 murine marrow cells showed peak pulmonary epithelial cell-specific mRNA expression in cell cycle phase G0/G1 when the vesicles were derived from irradiated lung tissue while the peak was in late G1/ early S phase when the vesicles were derived from nonirradiated lung. Vesicles were present in all types of differentiated marrow cells. Vesicles demonstrated a capacity to reverse radiation damage to marrow and gastrointestinal tissues of mice with the most impressive effect being on long-term engrafting stem cells [36]. The vesicles were shown to increase proliferation, decrease apoptosis and reverse double-strand

*DOI: http://dx.doi.org/10.5772/intechopen.86845*

**1.2 Hematopoietic stem/progenitor cells**

### *Stem Cells and Extracellular Vesicles: Biological Regulators of Physiology and Disease DOI: http://dx.doi.org/10.5772/intechopen.86845*

from red blood cells and platelets and felt to be essential cellular waste products [28, 29]. Subsequent work has characterized and subdivided these entities to size, density and morphology. Eventually two basic types of vesicles were defined by differential ultracentrifugation; exosomes and microvesicles. Exosomes derived from multivesicular bodies were from 30 to 100 nm in diameter while microvesicles derived from membrane blebbing were from 100 to 1000 nm in diameter. Other vesicular entities were also defined including apoptotic bodies. In general, there was much overlap between exosomes and microvesicular bodies and a meeting of investigators decided it might be best to simply term these as extracellular vesicles (EV) and then define their source and the conditions under which they were isolated. Vesicles were eventually isolated from virtually all bodily fluids and cells [26, 27]. Recent focus has been on the capacity of EV to restore injured tissue and treat disease. Initial work showed that vesicles could transfer protein and RNA while modifying the phenotype of cells and reversing disease in animal models.

Ratajczak et al. showed that embryonic stem cell derived microvesicles could reprogram hematopoietic progenitors by horizontal transfer of mRNA and protein delivery [30]. This was followed by work by Aliotta et al. [31–33] and Valadi et al. [34] showing RNA transfer and phenotype change in different experimental models. Further work has indicated that cellular phenotype change may be mediated by transfer of transcriptional activators possibly miRNA [35]. Vesicles from different sources are different but do contain some features of their originating cells. Vesicles contain protein, mRNA, miRNA, lipids and variably DNA, thus they represent complex bio machines with a tremendous range of potential phenotype altering messages.
