A Platform for Studying of the Three-Dimensional Migration of Hematopoietic Stem/Progenitor Cells

Abstract

Background:

Hematopoietic stem/progenitor cells (HSPCs) have the property to return to the bone marrow, which is believed to be critical in situations such as HSPC transplantation. This property plays an important role in the stemness, viability, and proliferation of HSPCs, also. However, most in vitro models so far have not sufficiently simulated the complicate environment. Here, we proposed a three-dimensional experimental platform for the quantitative study of the migration of HSPCs.

Methods:

After encapsulating osteoblasts (OBs) in alginate beads, we quantified the migration of HSPCs into the beads due to the physical environment using digital image processing. Intermittent hydrostatic pressure (IHP) was used to mimic the mechanical environment of human bone marrow without using any biochemical factors. The expression of stromal cell-derived factor 1 (SDF-1) under IHP was measured.

Results:

The results showed that the presence of OBs in the hydrogel scaffold initiate the movement of HSPCs. Furthermore, the IHP promotes the migration of HSPCs, even without the addition of any biochemical factors, and the results were confirmed by measuring SDF-1 levels.

Conclusion:

We believe this suggested three-dimensional experimental platform consisting of a simulated in vivo physical environment and encapsulated OBs should contribute to in vitro migration studies used to investigate the effects of other external factors.

Introduction

Hematopoietic stem/progenitor cell (HSPC) transplantation is widely used to treat hematological diseases, such as leukemia and other blood cell cancers [1, 2]. For successful transplantation, it is important to maintain or increase cell mobilization from the bone marrow (BM) throughout the blood circulation, as well as cell homing for return to the BM microenvironment [3–7]. The binding of HSPCs in their BM niche affects their stemness, viability, and proliferation [8]. Therefore, it is important to be able to study HSPC migration. Several experimental techniques have been introduced for this purpose because many more factors and environmental conditions are involved in the migration of HSPCs than in the migration of other cells. Sobkow et al. [9] attempted to regulate the migration of HSPCs by mimicking the endothelial lining of the BM using a Transwell, human umbilical vascular endothelial cells, and a hydrogel containing stromal cell-derived factor 1 (SDF-1). De Barros et al. [10] constructed a spheroid with mesenchymal stem cells (MSCs) or osteoblasts (OBs), which are involved in the homing of HSPCs in the BM, to study the movement of HSPCs by simulating the BM niche. However, Transwells cannot match the low stiffness and three-dimensional (3D) structure of the BM [11, 12], and the spheroid model requires a complicated analysis process. Moreover, few models that consider the in vivo mechanical environment have been developed. Therefore, we developed a simple model of the BM niche that uses a hydrogel scaffold and feeder cells, and examined its use for HSPC migration experiments.

Our model consists of alginate beads, OBs, and intermittent hydrostatic pressure (IHP). OBs in the BM are one of the major factors involved in the homing of HSPCs [7, 13]. Alginate beads with low stiffness and a 3D structure were used to mimic the organization of the BM [14]. IHP was introduced to mimic the mechanical environment that OBs experience in the BM niche [15]. In vivo, mechanical stimulation in the BM is an important determinant of the fate of stem cells, and helps maintain homeostasis in the BM [15].

The intramedullary pressure is determined by the blood flow into and out of the bone, and is about one-quarter of the systemic blood pressure [15]. This phenomenon was taken into account.

To quantify the migration of HSPCs in the vicinity of OBs in a bead, the number of migrated HSPCs was counted using digital image processing. The changes in SDF-1 levels [16–19] in the medium were also measured to assess the changes in migration due to IHP.

In summary, the proposed platform did not use any biochemical factors, and instead used only a mechanical factor involved in the migration of HSPC. This approach has not been considered in most related studies, although mechanical factors are very important. We believe that the platform will contribute to more systematic studies of the effects of various biochemical factors on the migration of HSPCs.

Materials and methods

Preparation of cells and alginate beads

Human OBs were obtained from PromoCell (Heidelberg, Germany), and HSPCs were obtained from STEMCELL Technologies (Vancouver, BC, Canada). The OBs were cultured in Osteoblast Growth Medium (Ready-to-use) supplemented with SupplementMix (PromoCell, Heidelberg, Germany) in an incubator at 37 °C and 5% CO₂. Cells from passage 5 were used for the experiments, and the medium was replaced every other day. HSPCs were cultured in StemSpan™ Serum-Free Expansion Medium II (SFEM II, STEMCELL Technologies, Vancouver, BC, Canada), supplemented with recombinant human stem cell factor (SCF; 50 ng/mL; ProSpec, Rehovot, Israel), recombinant human FMS-related tyrosine kinase 3 ligand (FLT3; 50 ng/mL; ProSpec, Rehovot, Israel), and recombinant human thrombopoietin (TPO; 20 ng/mL; ProSpec, Rehovot, Israel) under the same conditions as the OBs, except half of the medium was exchanged every other day.

The OBs were encapsulated in alginate beads to construct a 3D environment. Alginate solutions were prepared at three different concentrations (0.6, 1.2, and 2.4 wt.%) as described previously [20], and 1 × 10⁴ OBs were introduced into each bead using a 32G needle.

The biocompatibility of the alginate beads with different concentrations was confirmed using the LIVE/DEAD^® Viability/Cytotoxicity Kit (Thermo Fisher Scientific, Waltham, MA, USA). For this, beads containing OBs were cultured for 3 days and then dissolved in 55 mM trisodium citrate dihydrate (Junsei Chemical, Tokyo, Japan) in 150 mM NaCl. After being washed with Hanks’ Balanced Salt Solution (HBSS; Sigma-Aldrich, St. Louis, MO, USA), the cells were treated with LIVE/DEAD assay reagents and incubated at 37 °C for 30 min and then rinsed with HBSS. Finally, the state of the cells was identified using fluorescence microscopy (Carl Zeiss, Oberkochen, Germany).

Establishing the 3D co-culture system

To establish the co-culture system, each alginate bead containing OBs was placed in a round-bottom 96-well plate with 20 μL of StemSpan™ SFEM II containing 2,000 HSPCs (Fig. 1A).

Fig. 1.

3D co-culture system and process used to determine the numbers of HSPCs and OBs. A Schematic drawing of the co-culture system. B Configuration of the IHP stimulation system. C Stained image. D Images showing extraction of the red or green layer. E Binary image used for detecting cells (scale bar = 200 μm)

Prior to co-culture, the OBs and HSPCs were labeled with colored cell trackers to distinguish the two cell types in the alginate beads when HSPCs migrated into the bead. The OBs were treated with 5 μM CellTracker™ Green CMFDA (Thermo Fisher Scientific, Waltham, MA, USA) in Osteoblast Growth Medium, and the HSPCs were treated with 5 μM CellTracker™ Red CMTPX (Thermo Fisher Scientific, Waltham, MA, USA) in StemSpan™ SFEM II at 1 × 10⁶ cells/mL, and all were incubated at 37 °C for 30 min. The OBs and HSPCs were washed with HBSS and then with Iscove’s Modified Dulbecco’s Medium (IMDM; Thermo Fisher Scientific, Waltham, MA, USA) with 1% Albumin Bovine Fraction V (BSA; MP Biomedicals, Santa Ana, CA, USA). The OBs were labeled with the corresponding tracker before being encapsulated in the beads.

Mechanical stimulation

IHP was applied using a commercial bioreactor (ACBC-100; AnyCasting, Gimhae-si, Korea) (Fig. 1B) at a pressure of 20 kPa for 2 min [15, 21, 22], followed by no pressure for 4 min. The bioreactor was run for up to 17 h. One group was not stimulated for the last 5 h to examine the persistence of the stimulus effect.

Counting of the number of HSPCs migrating into the beads

The beads recovered at each time point were washed with 102 mM CaCl₂ and 150 mM NaCl, fixed with 4% paraformaldehyde for 10 min at room temperature, and rinsed with 150 mM NaCl. Single beads were placed on a confocal microscope (LSM 800; Carl Zeiss, Oberkochen, Germany) to obtain images of the migrated HSPCs. The red or green layer was extracted from the images using MATLAB ver. R2017b (MathWorks, Natick, MA, USA), and the number of cells was counted through image processing (Fig. 1C–E).

SDF-1 assay

The concentration of SDF-1 in the medium was measured. Medium was collected at 12 and 17 h and centrifuged at 300×g for 10 min. The SDF-1 concentration in the supernatant was measured with a Human CXCL12/SDF-1α Quantikine^® ELISA Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Briefly, 100 μL of standard and sample solutions were incubated at room temperature for 2 h. After washing with buffer, SDF-1α conjugate solution was added, and the mixture was incubated for 2 h at room temperature. Then, the substrate and stop solutions were added. The optical density was determined at 450 nm using a Multiskan EX (Thermo Fisher Scientific, Waltham, MA, USA), and the SDF-1 concentration was determined using a standard curve.

Statistical analyses

Data from four independent experiments were analyzed with the t test and one-way analysis of variance using IBM SPSS Statistics (ver. 25.0; SPSS, Chicago, IL, USA). The level of significance was set at p < 0.05.

Results

Determination of the appropriate concentration of alginate beads

The encapsulated OBs were subject to LIVE/DEAD staining to determine the optimal bead concentration. Fluorescence microscopy observations after culturing the OBs in the beads for 3 days indicated that number of dead cells increased with the bead concentration (Fig. 2A). The numbers of live and dead cells were counted using MATLAB, and the results were expressed as the ratio of living cells to the total number of cells. Both the 0.6 and 1.2 wt.% concentrations proved suitable (Fig. 2B). We selected alginate solution with 1.2 wt.% concentration for this study because of its ease of handling.

Fig. 2.

OB viability on day 3. A Typical images of OBs in alginate concentrations of 0.6, 1.2, and 2.4 wt.% treated with LIVE/DEAD reagent (scale bar = 200 μm). B Ratio of living cells to total cells (*p < 0.05)

Change in the migration of HSPCs with mechanical stimulation

To confirm that OBs induce the migration of HSPCs in the proposed system, we checked whether HSPCs migrated into the beads containing encapsulated OBs after 5, 8, 12, and 17 h. Figure 3A shows typical images of migration when the beads contained OBs. In the absence of OBs, only a negligible number of HSPCs migrated (Fig. 3B).

Fig. 3.

Effects of OBs and IHP stimulation on the migration of HSPCs. A Representative images of HSPCs (red, left) in alginate beads containing OBs (green, right) at 5, 8, 12, and 17 h (scale bar: 200 μm) without mechanical stimulation. B Numbers of HSPCs in alginate beads with or without OBs (n = 5). C Numbers of HSPCs quantified by OBs under IHP (n = 9) (*p < 0.05)

Figure 3C shows the ratio of the numbers of HSPCs to OBs in beads, showing that mechanical stimulation promoted the migration of HSPCs. There were two stimulated groups: one stimulated for 12 h and the other for the entire 17 h. The migration observed in the two stimulated groups did not differ after 17 h, implying that the effect of stimulation persisted for a considerable period.

Expression of SDF-1

Many factors are involved in cell migration, including SDF-1 [16–19]. The SDF-1 concentration used in the other groups was based on the SDF-1 concentration measured at 12 h in the static group (Fig. 4). The SDF-1 concentration in the medium had increased at 17 h, and was higher when mechanical stimulation was applied. Therefore, although there was a correlation between the migration of HSPCs and SDF-1 expression, the expression of SDF-1 alone cannot explain the entire migration phenomenon.

Fig. 4.

Concentration of SDF-1 in medium measured at 12 and 17 h (n = 3–4)

Discussion

In this study, we constructed a simple model of BM to enable the study of HSPC migration in vitro. First, alginate beads with OBs were used to simulate the BM niche as they have low 3D stiffness [14]. OBs were used as a representative BM cell type as they play important roles in the homing [7, 13] and survival [23, 24] of HSPCs by producing many factors.

First, we checked the biocompatibility of the alginate beads with different concentrations. Both the 0.6 and 1.2 wt.% had higher viability than 2.4 wt.%. However, the alginate beads made with alginate solution concentration of 0.6 wt.% could not maintain the 3D shape for experimental time. And the OBs in the beads showed the tendency that they escaped from the beads as the experiment progressed. Therefore, we chose 1.2 wt.% concentration of alginate solution for this experiment.

The presence of OBs in the beads influenced the movement of HSPCs, as expected, and no HSPCs were detected in beads without OBs. The number of HSPCs in the alginate beads increased with time, and the survival and migration ability of the HSPCs was maintained throughout the experiment. This system, consisting of only a hydrogel scaffold and OBs, appears to be a useful, simple BM model for HSPC migration studies.

Most migration studies have examined the effects of the surrounding cells or biochemical environment, whereas few have examined mechanical stimulation. In particular, IHP is known to have a significant effect on the OBs and HSPCs used in this experiment. OBs exposed to IHP stimulation secrete various cytokines and factors [25, 26], and we confirmed that IHP positively affects the maintenance and proliferation of HSPCs in previous studies [21, 22]. In studies that utilize mechanical stimuli, it is important to determine the optimal extent and pattern of the stimuli. We selected a pressure of 20 kPa for 2 min followed by a 4-min rest, based on prior studies [21, 22] and a preliminary experiment (data not shown). The IHP mimicked the mechanical environment of the BM and promoted the movement of HSPCs. The number of HSPCs migrating into the alginate beads was significantly higher under IHP stimulation. This suggests that IHP promotes the migration of HSPCs, even without the addition of biochemical factors, and further demonstrates the importance of simulating physical factors when constructing in vitro BM models. However, there was no significant difference in the results of the groups stimulated for 12 or 17 h, suggesting that the effects of IHP persist following stimulation.

SDF-1 is expressed in OBs in the BM [23, 24] and guides the return of HSPCs to the BM [16–19]. SDF-1 is believed to the major factor that regulates HSPCs migration by combining with C-X-C chemokine receptor type 4 (CXCR4) expressed by HSPCs and induce the rolling and homing of HSPCs in vivo [27]. Kang et al. and Kim et al. [21, 28] confirmed that IHP stimulation can promote the secretion of SDF-1 in MSCs. We also found that the expression of SDF-1 was elevated under IHP stimulation. However, many factors are involved migration, and this result may be partly due to the increased migration under IHP. Since few samples could be collected in one experiment, several independent experiments were performed and each showed the same tendency. However, HSPC homing involves factors other than SDF-1, such as the BM extracellular matrix (ECM) [29, 30]. Further studies should examine the interrelationships of BM ECM, collagen I, fibronectin, and SDF-1.

In conclusion, this study proposed a simple in vitro BM biomimetic system that can be used to study HSPC migration. The ability of IHP to simulate the physical conditions in the BM related to the movement of HSPCs was confirmed by measuring SDF-1 expression. The IHP stimulation affected OBs to induce increase of SDF-1 expression. This platform is believed to be useful for research conducted to investigate the effects of other external factors in HSPC migration.

Compliance with ethical standards

Conflict of interest

All authors have no conflict of interest to declare.

Ethical statement

There are no animal experiments carried out for this article.