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The role of reactive oxygen species (ROS) in hematopoietic

stem cells engraftment

Minji Choi , Mina Boo , Woo Yong Park , Gahee Song , Ja Yeon Park , Se Jin Jung , Jinbong Park , Jae-Young Um ,
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Hyun Jeong Kwak ¹*

1 Department of Natural Science, College of Convergence and Integrated Science, Kyonggi University, Suwon 16227,

Korea 2 Department of sience of Korean Medicine, Graduate school, Kyung Hee University, Seoul 02447, Korea

3 Department of pharmacology, Collage of Korean Medicine, Kyung Hee University, Seoul 02447, Korea

ABSTRACT RESULTS

Hematopoietic stem cell transplantation (HSCT) can be curative for hematological and

immunological diseases. The success of HSCT is dependent on their ability of homing and

engraftment to the hematopoietic stem cells (HSCs) of recipients. Reactive oxygen species (ROS)

is traditional second messengers in many cell type including HSCs, however high levels of ROS

exhausted hematopoietic stem cells (HSCs). Therefore, in this study we evaluated whether Experimental setup for transplantation

maintaining a low levels of ROS in HSCs may augment the engraftment efficiency of HSCs.

Transplantation of donor BM cells (CD45.1+) into recipients mice (CD45.2+) with a GSH

biosynthesis inhibitor, buthionine sulfoximine (BSO) displayed a significant increase in HSC

engraftment, and this was perturbed in the presence of ROS scavenger, N-acetyl-L-cysteine

(NAC). In comparison with control mice, BSO-treated recipients displayed a 9.5 fold increase in

hematopoietic engraftment. Specifically, engraftment efficiency was lower in 25 mg/kg BSO-

treated mice than in 5 mg/kg BSO. Moreover, granulocyte/macrophage progenitors (GMPs)

populations was increased in recipient mice, suggesting that enhancement of cell differentiation

and proliferation of myeloid progenitor cells. Taken together, our results suggest a diverse role

of ROS that regulates HSC function. Therefore, low ROS has the potential as a therapeutic

approach in HSCT.

PURPOSE

•To investigate whether ROS improve HSC engraftment and homing following BM transplant. Therefore, we BSO treatment (5 mpk, ip)

asked if this beneficial effect of ROS on HSCT is dependent on ROS concentration , differentiation and

proliferation of myeloid progenitor cells.

MATERIALS AND METHODS

Flow cytometry and antibodies

The antibodies used for flow cytometry included the following:

APC conjugated lineage markers specific for CD3e (145-2C110), CD4 (RM4-5), CD8a (53-6.7), CD11b(M1/70),

B220 (RA3-6B2), GR-1 (RB6-8C5), and Ter119 (TER119). Other antibodies included PC-Cy7- or FITC-conjugated

Sca-1 (D7), APC-Cy7-conjugated c-kit (2B8), APC conjugated CD45.2 (104), PE-Cy5-conjugated CD3e (145-

2C11), PE-conjugated CD45.1 (A20), PE-conjugated CD16/32 (93), FITC-conjugated CD34 (RAM34). Unstained

cells were used as negative control to establish the flow cytometer voltage setting, and single-color positive

controls were used for adjustment of the compensation. The flow cytometric data were acquired using

FACScalibar, and raw data were analyzed with Flowjo software (Treestar Inc).

BM cell transplantation

Age-matching C57BL/6 and CD45.1 mice were purchased from Jackson Laboratories. Donor whole ROS leads to increase progenitor cell proliferation in the bone marrow

BM cells (WBM) were prepared by spinning femurs and tibias under sterile conditions, and red

blood cells were lysed using ACK lysing buffer. LK progenitor cells were sorted using a FACS AriaII

equipped with FACSDiva software (BD Bioscience). The transplantation was conducted using non-

irradiated WT (CD45.2) recipient mice. The donor whole BM (CD45.1, 3 × 10 ) and LK cells
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(CD45.1, 2 × 10 ) were transplanted into each non-irradiated WT (CD45.2) recipient mouse via
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tail vein injection. To increase the efficiency of engraftment, we administered BSO into recipient

mice every 2 days for 1 week. All animal manipulations were conducted in accordance with the

Animal Welfare Guidelines of the Kyung Hee university.

Granulocyte and Monocyte Colony-Forming Unit Assays

Bone marrow cells (2 × 10 ) from WT were seeded in semisolid Methocult GF M3534 medium
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containing rmSCF, rmIL-3, and rhIL-6 for detection of CFU-GM (Stem Cell Technologies). L- (LK cell transplantation) (Whole BM cell transplantation)

butionine-sulfoxamine (BSO, Sigma-Aldrich) was added to methylcellulose media at the indicated

concentrations at the time of plating.

Homing assay

BM cells derived from wild type (CD45.1, 2x10 ) were re-suspended in culture medium and add cell
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tracking

fluorescent dyes: CMFDA (green; 10 mil/ml conc. 5 uM) for 15 min at 37 C shaker. Cells were wash
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with 4 ml complete medium and spin down. And then re-suspend cells injected recipient mice

(CD45.2) by iv. After 16 hr, CD45 ab were iv injected into the recipient mice. Mice were sacrificed

within 2 min. And then bone marrow cells were isolated and stained with specific ab against lin-,

c-kit and sca-1. Facs and the homing efficiency of progenitor cells were analyzed.

BACKGROUND

(LK cell transplantation) (Whole BM cell transplantation)

CONCLUSIONS

In conclusion, we provide evidence that ROS enhances engraftment efficiency at a low concentration

after whole BM or LK cell transplantation. Moreover, these improvement on engraftment was

associated with GMP progenitor and GR-1+ cell proliferation, suggesting that ROS may be useful in

manipulation of HSC transplantation.

REFERENCES

1.Kwak HJ, Liu P, BajrAMI b, Xu Y, Park SY, Nombela-Arrieta C, Mondal S, Sun Y, Zhu H Luo HR (2015). Myeloid

cell-derived reactive oxygen species externally regulate the proliferation of myeloid progenitors in

emergency granulopoiesis. Immunity 42:159-71

2.Zhu H, Kwak HJ, Liu P, Bajrami B, Xu Y, Park SY, Nombela-Arrieta C, Mondal S, Kambara H, Yu H, Chai L,

Silberstein LE, Cheng T, Luo HR Reactive oxygen species-producing myeloid cells act as a bone marrow

nitch for sterile inflammation-induced reactive granulopoiesis (2017). J Immunol. 198:2854-2864.

ACKNOWLEDGEMENT

This work is supported by the National Research Foundation of Korea (NRF) grant funded by the

Korea government (NRF-2021R1A2C1012532)