A Novel View of Bone Marrow As a “stem Cell sensor” of Tissue/Organ Damage -Evidence That in Vivo Exposure to the Neurotoxin Kainic Acid (KA) Induces Proliferation and Neural Specification of Developmentally Early Stem Cells Directly in Bone Marrow Before They Are Mobilized Into Peripheral Blood

It is well known that various stem cells become mobilized into peripheral blood (PB) in response to tissue/organ injuries (e.g., heart infarct, stroke, or bleeding); however, the data on the immediate response of stem cells in BM during organ injuries are somewhat limited. We and others have demonstrated the presence of developmentally early stem cells in BM that we have named very small embryonic-like stem cells (VSELs). These Oct-4⁺SSEA-1⁺Sca-1⁺Lin^–CD45^– cells are kept quiescent in BM in the G₀ phase of the cell cycle by erasure of the somatic imprint in the differentially methylated regions (DMRs) of some crucial paternally imprinted genes, (Igf2-H19, RasGRF1, and p57^Kip2) that regulate proliferation of embryonic stem cells (Leukemia 2009;23:2042). These cells are mobilized into peripheral blood, for example, during heart infarct (J Am Coll Cardiol 2009;6:1–9.), stroke (Stroke 2009;40:1237–44.), or skin burns (Stem Cell Rev. 2012;8:184–94.).

We hypothesized that this population of BM-residing, small, quiescent, pluripotent cells should be able to respond to organ injury induced by a known neurotoxin, kainic acid (KA), in a brain damage model. We hypothesized that these quiescent cells would began to proliferate, expand, and become specified into the neural lineage.

C57Bl6 mice were injected with increasing doses of KA and at various time intervals mice were sacrificed to harvest BM, PB samples, and brains for analysis. Brain damage was confirmed by histological analysis. The number of Sca-1⁺Lin^–CD45^– VSELs and Sca-1⁺Lin^–CD45⁺ HSPCs was evaluated in BM and PB by FACS. The cell cycle status of VSELs and HSPCs was evaluated by FACS in cells isolated from mice that received bromodeoxyuridine (BrdU) after KA injection. By employing RQ-PCR, we also measured the expression of genes that regulate stem cell pluripotency (Oct-4, Nanog, Sox2, and Rex1) and regulate neuronal development (Nestin, βIII-tubulin, Olig1, Olig2, and GFAP). The expression of these genes was subsequently confirmed in sorted cells by immunohistochemical staining. The numbers of clonogenic CFU-GM and BFU-E progenitors residing in BM and circulating in PB were tested in methylcellulose cultures.

We found that 12 hrs after administration of KA (25 mg/kg bw) quiescent VSELs residing in BM enter the cell cycle: ∼2 ± 1% for control vs. 37 ± 6% for KA-treated cells. Interestingly, at the same time we did not observe significant changes in the proliferation rate of HSPCs (15±5% for control vs. 17±4% for KA-treated cells). The elevated number of VSELs in the cell cycle remained detectable for a few days and returned to control values (∼2%) after 1 week after KA administration. Furthermore, an increase in the number of cycling VSELs correlated with an increase in expression of pluripotent markers, according to RQ-PCR analysis. In parallel, 48 hrs after KA administration we observed the release from BM into PB of Sca-1⁺Lin^–CD45^–VSELs highly enriched for mRNAs characteristic of neural differentiation. Interestingly, while we observed a significant increase in VSEL number in BM and PB after KA-induced brain damage, no significant changes were observed for both BM-residing and circulating HSPCs.

For the first time, we provide evidence that the compartment of developmentally early stem cells residing in BM responds robustly to brain damage induced by a neurotoxin. This effect seems to be specific for VSELs, as no significant changes were observed for HSPCs. The kinetics of changes in BM revealed that BM VSELs enter the cell cycle and, after they become specified into the neural lineage, egress from BM and enter the PB. Thus, our data provide novel evidence that developmentally early stem cells in BM “sense” the damage to brain tissue and respond to this type of organ injury. In parallel, we are studying the specificity of the response of BM-residing VSELs and HSPCs to other types of organ damage, such as heart infarct and acute limb ischemia.

Ratajczak:Neostem Inc: Member of SAB Other.