Effect of simulated microgravity conditions of hindlimb unloading on mice hematopoietic and mesenchymal stromal cells

Abstract Conditions in space, such as microgravity, may affect the hematopoietic and bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) of astronauts. However, to date, few detailed phenotype change data about the different type of hematopoietic cells have reported. In this study, C57BL/6 mice were randomly divided into two groups: a control group (control) and a hindlimb suspension group (treated). After four weeks of hindlimb suspension, we found that this simulated microgravity (sµg) condition could increase the percentage of monocytes and macrophages and decrease the percentage of B lymphocytes and mature red cells in bone marrow. The percentage of B lymphocytes in the spleen and the red blood cell count in peripheral blood also decreased, consistent with the response of bone marrow. The cytoskeleton in the BM‐MSCs was disrupted. The expression levels of hematopoietic‐related genes, such as fms‐like tyrosine kinase‐3 ligand, granulocyte‐macrophage colony stimulating factor, interleukin‐3, and adipogenic differentiation associated genes, leptin and proliferator‐activated receptor γ type 2, were upregulated under sµg conditions. These results indicated that simulating microgravity can affect the phenotype of certain types of hematopoietic cells and the morphology and gene expression pattern of BM‐MSCs.


| INTRODUCTION
The environment in space includes exposure to various forces, including microgravity, radiation, alterations in circadian rhythm, and extreme temperatures. Studies of humans and animals that have experienced space flight (SF) have shown that many tissues and organ systems display some measurable changes. In some cases, these changes are minor, and their relevance to astronaut health has not been determined (Globus and Morey-Holton, 2016). Microgravity is known to enhance cancer risk and influence biological systems, including bone, muscle, heart, and brain (Ulbrich et al., 2014). Human SF missions have resulted in some hematological anomalies. These effects of microgravity on hematological function have drawn the attention of researchers to those effects on the health of astronauts from the outer space.
Many studies have shown that microgravity dramatically affects cell morphology, proliferation, differentiation, and signal transduction. Currently, the influence of microgravity on isolated cells is being studied in vitro (Y. N. Zhang et al., 2018). However, the results obtained from in vitro experiments do not represent the in vivo situations. Considering that it is not practicable to gather enough tissue samples from astronauts for a thorough investigation, hindlimb suspension has been developed as a means to simulate the effects of microgravity, enabling studies of the effects of microgravity on the biology and behavior of mice (Ulbrich et al., 2014).
Bone marrow mainly includes two types of cells with respect to their origin-hematopoietic and mesenchymal (Ozcivici, 2013). The morphology, phenotypes, and differentiation of these cells may all be affected by microgravity. Davis' group investigated the in vitro effects of SF on CD34 + hematopoietic cell proliferation and differentiation during the space shuttle missions  and . (Blaber, Sato, & Almeida, 2014;Davis et al., 1996). Domaratskaya, Michurina, et al. (2002) analyzed the effect of SF on the clonogenic hemopoietic cells numbers of newts. Sotnezova, Markina, Andreeva, and Buravkova (2017) evaluated the content of myeloid stem colony-forming unit (CFU) in mice bone marrow karyocytes after a 30-day Bion-M1 pace flight and observed a significant decrease in the number of erythroid progenitors, including common myeloid precursor after the SF. Markina, Andreeva, Andrianova, Sotnezova, and Buravkova (2018) also evaluated the effects of 30-day SF on biosatellite on the mononuclear cells (MNCs) of murine bone marrow progenitors and discovered the total hematopoietic CFU number decreased. Huang et al. (2009) studied the differentiation of rat bone marrow mesenchymal stem cells in vitro under simulated microgravity (sµg). Markina, Andrianova, Shtemberg, and Buravkova (2018) studied the effect of 30-day hindlimb unloading (HU) on the clonogenic and differentiation potential of bone marrow stromal progenitors in mice. They found clonogenic and differentiation activity of stromal cells decreased after unloading.
Above mentioned results from real and sµg, long or short time, including rodent experiments, mainly focused on the cell count, function and morphology. Thus, detailed phenotype data describing these changes also need further study.
To observe whether sµg, which specifically affects the muscular and skeletal systems in rodents, can also affect other systems, such as the hematopoietic system, and in what degree they will be affected, hindlimb suspension was used in this study to evaluate the effects of microgravity simulation on hematopoietic and mesenchymal stromal cells (MSCs) in vivo.
2 | MATERIALS AND METHODS 2.1 | Hindlimb suspension 6-8-week-old C57BL/6N mice, with body weights of 20-22 g, were purchased from Vitalriver Corporation (Beijing, China) and acclimatized for 1 week in an air-conditioned room at a temperature of 23±2°C with 12 h/12 hr light-dark illumination cycles and humidity at 45-50%. The mice were randomly divided into a control group (control, n = 7) and a hindlimb suspension group (treated, n = 14).
Treated mice were subjected to hindlimb suspension according to the method of Morey-Holton and Globus (1998) andY. N. Zhang et al. (2018). Briefly, hindlimbs were suspended at a 30°angle using a paper clip, adhesive tape, and a metal tail harness. The tail harness was attached to a swivel buckle mounted on a guide wire running the length of the cage via a metal chain. Using this setup, mice were able to access all areas of the cage. The hindlimb suspension was main-

| Analysis of the surface markers of hematopoietic cells by flow cytometry
Femoral bones were sampled, the bone marrow was flushed out by drawing and expelling with a syringe and single cells were collected.
Spleens were collected and grounded to obtain a single splenocyte.
Heparin anti-coagulated peripheral blood samples were collected from each group. The splenocyte and peripheral blood cells were lysed by 1×RBC lysis buffer (Beyotime, Shanghai, China) for 10 min.
Surface markers for each cell type were quantified by flow cytometry and the cell number used for analysis was 1×10 6 /tube.
Monoclonal antibodies APC-Cy7-Lin − , PE-c-kit, and PE-Cy7-sca-1 were used for the detection of LSK cells in bone marrow. After washing, cell nuclei were counterstained with diamidinophenylindole for 5 min. Cells were viewed and photographed by using a confocal microscope (Ultra view VOX, PerkinElmer).

| Expression of hematopoietic promotion-and differentiation-related genes in mouse cultured BM-MSCs
Mice were sacrificed on Day 28 of microgravity simulation, and bone marrow were rinsed from femoral bone and cultured in α-MEM medium containing 10% FBS for about 12 days. To detect changes in the expression of hematopoietic promotion-and differentiation-related genes, cultured BM-MSCs total RNA was isolated with TRIzol reagent (Life Technologies, Carlsbad, CA).
We used 2 −ΔCt to represent the expression level. RT-qPCR was conducted in ABI 7500 Fast PCR System (Applied Biosystems, CA).

| Statistics
Data were analyzed using one-way analysis of variance statistical analysis. The data are expressed as the mean ± SEM (standard error of the mean). GraphPad Prism version 6 (GraphPad Software) was used for statistical analysis. Student's t-test was used to evaluate the statistical significance. In all of the analyses, p < .05 was considered to be significant.

| RESULTS AND DISCUSSION
The rodent HU model is thought to mimic microgravity conditions and has been applied to characterize the effects of SF on integrated physiological, organ-specific, and mechanistic responses (Globus and Morey-Holton, 2016). In this study, we used an HU model ( Figure S1) to observe the effects of sµg on hematopoietic cells and BM-MSCs and found that some characteristics and/or morphology of these cells were changed.

| Simulated microgravity decreased RBC counts in peripheral blood
To evaluate the effect of sµg on blood cell count in our study, mice underwent hindlimb suspension, and peripheral blood samples were taken at different time points. We did not observe Ly6G is expressed predominantly on neutrophils but can also be expressed on differentiating premonocytes and other cell types (Ortega et al., 2009). Approximately 41.8 ± 4.7% of bone marrow cells from animals exposed to sµg were positive compared with 34.1 ± 4.4% (p < .01) for control mice (Figure 2f).  Ly6C is absent in cells of erythroid lineage and completely lost by cells upon final maturation into macrophages (Ortega et al., 2009). Overall, 19.3 ± 1.7% of bone marrow cells from sµg-exposed animals were positive compared with 15 ± 1.8% (p < .01) for control mice (Figure 2g).
Vacek, Michurina, Serova, Rotkovska, and Bartonickova's (1991) work showed that compared to cells cultured under normal gravity, the numbers of rat bone marrow cells, granulocytes, macrophages, and hematopoietic progenitor cells were significantly lowered when cultured onboard the Cosmos-2044 Biological Experimental Satellite. But Davis et al.'s (1996) work demonstrated that microgravity cultures could accelerate maturation/differentiation toward the macrophage lineage.
The percentage of B220 + (expressed on the B lymphocyte) lymphocytes in our study decreased (Figure 2i) after sµg. The difference between the two groups achieved statistical significance (10.9 ± 2.6% vs. 20.7 ± 3.5%, p < .01). HU can alter the quantity, distribution, and lymphopoiesis of lymphoid cells in bone marrow (Lescale et al., 2015). Ichiki et al. (1996) observed that the numbers of lymphoid progenitor cells isolated from rats undergoing SF decreased. Other data suggest that mature B cells were significantly suppressed, accompanied by increased natural killer cells in rodents exposed to SF ). Domaratskaya et al. (2002) verified that the number of RBCs also decreased. Using FACS, we elucidated that sµg decreased mature erythrocyte counts in bone marrow but not basophilic normoblasts, polychromatic normoblasts, acidophilic normoblasts, or reticulocytes (Figure 2j-n). This is the first report about the effect of sµg on the different developmental stage of bone marrow RBCs. Davis et al. (1996), Lo Celso et al. (2009), Plett, Abonour, Frankovitz, and Orschell's (2004 study showed that erythroid progenitor cell numbers and erythroid development reduced at microgravity/modeled microgravity. These reports combined our results could explain why humans subjected to periods of microgravity develop anemia.
In short, our study showed that sµg increased the percentages of macrophages, granular leukocytes, and monocytes and slightly in-

| Phenotype of spleen and peripheral blood lymphocytes
In this study, spleens were collected and lymphocytes were separated at Day 28 of microgravity simulation. Phenotypes were analyzed by FACS and the results showed that sµg decreased the percentage of B220 + lymphocytes (Figure 3b), which was in accordance with the results from bone marrow, but no effect was seen on CD4 + T lymphocytes, CD8 + T lymphocytes, or the CD4 + / CD8 + ratio (Figure 3c-e).
Peripheral blood mononuclear cells were separated, and CD3, CD4, CD8 (Figure 4a-d) and Treg cells (Figure 2e,f) were detected by FACS. We observed that sµg had no effect on the phenotype of peripheral blood T lymphocytes.
Microgravity may affect the phenotypes of splenocytes and peripheral lymphocytes. The numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood of rats on SF decreased compared to rats under normal gravity, but spleen lymphocytes did not differ (Ichiki  , 1996). Other studies also showed that the number of T cells in peripheral blood was reduced during SF in both humans and rodents Ichiki et al., 1996). But in our study, populations of circulating T lymphocytes of treated mice have not significantly change compared with that of control mice (Figure 4) 3.4 | Effects of microgravity simulation on the cytoskeleton, differentiation-related genes and hematopoietic growth factor expression in cultured BM-MSCs We used FACS to detect the phenotype of cultured BM-MSCs and discovered that they expressed the phenotype of MSCs, CD29 and Sca-1, and didn't express the phenotype of HSCs (CD34) and vascular endothelium (CD31; Figure S2). force-insensitive cells, such as adipocytes (Huang et al., 2009).
hMSCs in a rotating wall vessel (RWV) bioreactor do not express osteogenic fate genes, such as alkaline phosphatase (ALP), collagen-I (COL-1), osteonectin (OS), and Runx2, or express them at lower levels. In contrast, the expression of lipogenic fate genes is upregulated under sµg conditions (Zayzafoon, Gathings, & McDonald, 2004;Zheng et al., 2007). These results showed that sµg can affect the differentiation ability of MSCs in vitro and in vivo, which are similar to that of our above mentioned findings.
The mechanism by which microgravity influences MSCs differentiation is thought to be related to the cytoskeleton, which is involved in intercellular signaling (Cau and Hall, 2005;Hosu, Mullen, Critser, & Forgacs, 2008). Studies have shown that microgravity can change MSCs differentiation potential by suppressing microfilament formation and RhoA activity or by increasing phosphorylation of p38 MAPK in these cells (Meyers, Zayzafoon, Douglas, & McDonald, 2005).

| CONCLUSIONS
Simulating microgravity could influence the percentages of certain