Microfluidic Separation of Canine Adipose-Derived Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are potential treatments for a variety of veterinary medical conditions. However, clinical trials have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. Identification and characterization of subpopulations within MSC cultures may improve those outcomes. Therefore, the functional heterogeneity of different-sized subpopulations of MSCs was evaluated. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization, as well as to evaluate the impact of culture conditions on their functional heterogeneity. We found that culture-expanded canine Ad-MSCs comprise distinct subpopulations: larger MSCs (mean diameter of 18.6 ± 0.2 µm), smaller MSCs (mean diameter of 15.3 ± 0.2 µm), and intermediate MSCs (mean diameter of 16.9 ± 0.1 µm). In addition, proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. We observed that larger MSCs proliferate more slowly, senesce at earlier passages, and are inclined to differentiate into adipocytes compared with smaller MSCs. Most importantly, these size-dependent functions are also affected by the presence of serum in the culture medium, as well as time in culture. Cell surface staining for MSC-specific CD44 and CD90 antigens showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs. Finally, transcriptome analysis showed differential gene expression between larger and smaller subpopulations of MSCs. Larger MSCs expressed genes involved in cellular senescence such as cyclin-dependent kinase inhibitor 1A and smaller MSCs expressed genes that promote cell growth [mechanistic target of rapamycin 1 (mTORC1) pathway] and cell proliferation [myelocytomatosis (myc), e2f targets]. These results suggest that different subpopulations of MSCs have specific properties. Impact statement Clinical trials of mesenchymal stromal cells (MSCs) from veterinary species have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization. Proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. Cell surface staining for MSC-specific cell surface markers showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs.

A serum-free medium formulation efficiently supports isolation and propagation of canine adipose-derived mesenchymal stem/stromal cells.

Medium containing Fetal Bovine Serum (FBS) provides a supportive environment for isolation and expansion of mesenchymal stromal/stem cells (MSCs); however, the inherent variability of FBS may contribute to inconsistencies in cell growth and yield between batches of stem cell products. For this reason, we set out to develop a serum-free medium capable of supporting the in vitro expansion of MSCs. First a naïve serum-free medium was formulated by Sato's approach. Once it was established that the naïve serum-free medium supported the expansion of canine adipose-derived MSCs (Ad-MSCs), the serum-free medium was optimized by addition of growth factors. Combinations of growth factors were chosen and compared by their effect on cell proliferation and colony formation. Growth characteristics of canine adipose-derived MSCs cultured in the serum-free medium were comparable to those cultured in standard FBS containing medium. In addition, cell surface marker expression and differentiation potential of serum-free and FBS-based cultures were also comparable. However, a commercial serum-free medium developed for human MSC culture did not support growth of canine Ad-MSCs. In summary, canine Ad-MSCs isolated and cultured in serum-free medium retained the basic characteristics of MSCs cultured in FBS containing medium.

Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24p3.

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.

A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production.

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. Here we report cloning of the 24p3 cell-surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo either iron uptake or apoptosis, dependent upon the iron content of the ligand: Iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis. Intracellular iron delivery blocks Bim induction and suppresses apoptosis due to 24p3 addition or IL-3 deprivation. We find, unexpectedly, that the BCR-ABL oncoprotein activates expression of 24p3 and represses 24p3R expression, rendering BCR-ABL(+) cells refractory to secreted 24p3. By inhibiting BCR-ABL, imatinib induces 24p3R expression and, consequently, apoptosis. Our results reveal an unanticipated role for intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL-induced myeloproliferative disease and its treatment.

Cloning and initial characterization of an alternatively spliced transcript encoded by the bovine herpes virus 1 latency-related gene.

Bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic sensory neurons of infected cattle. The latency-related (LR) RNA is the only abundantly expressed viral transcript in sensory neurons of latently infected calves. Wild-type expression of LR gene products is required for the latency-reactivation cycle in calves. LR RNA is alternatively spliced in trigeminal ganglia (TG) after infection of calves, suggesting that these alternatively spliced transcripts encode novel factors that regulate specific steps during latency. To begin testing whether these alternatively spliced transcripts have novel functions, the authors cloned a full-length cDNA identified in TG of calves at 7 days post infection (dpi) and compared the functions of this cDNA to the intact LR gene. As a result of splicing, the 7 dpi cDNA contains a novel open reading (ORF) comprised of OFR-2 fused to ORF-1. Overexpression of the 7 dpi cDNA inhibited the BHV-1 immediate-early transcription unit 1 (IEtu1) promoter and the herpes simplex virus type 1 ICP0 promoter. Conversely, the 7 dpi cDNA stimulated the LR promoter in transiently transfected cells. A plasmid containing the LR gene had little effect on IEtu1 or LR promoter activity, indicating that the 7 dpi cDNA has novel functions.

Transcriptional program of apoptosis induction following interleukin 2 deprivation: identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor.

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.

Inhibition of apoptosis by ATFx: a novel role for a member of the ATF/CREB family of mammalian bZIP transcription factors.

The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Here we report that expression of a specific mouse ATF gene, ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. We also show that 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression of ATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, our results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein.