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Transplantation of Donor–Recipient Chimeric Cells Restores Peripheral Blood Cell Populations and Increases Survival after Total Body Irradiation-Induced Injury in a Rat Experimental Model

Maria Siemionow
Maria Siemionow
, 
Małgorzata Cyran
Małgorzata Cyran
, 
Katarzyna Stawarz
Katarzyna Stawarz
, 
Lucile Chambily
Lucile Chambily
 e 
Krzysztof Kusza
Krzysztof Kusza
   | 23 mag 2024
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Article Category: Original
Pubblicato online: 23 mag 2024
Pagine: -
Ricevuto: 26 feb 2024
Accettato: 11 apr 2024
DOI: https://doi.org/10.2478/aite-2024-0009
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© 2024 Maria Siemionow et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig 1.

Experimental model of TBI for rats. Schematic image of application of the J.L. Shepherd, Model 143-68, employing Cs-137 radionuclide as the radiation source, with a metal cylinder designed for irradiation of the entire rat’s body. A Lewis experimental rat is placed within a specially adapted metal cylinder designed for the procedure of whole-body irradiation. TBI, total body irradiation.
Experimental model of TBI for rats. Schematic image of application of the J.L. Shepherd, Model 143-68, employing Cs-137 radionuclide as the radiation source, with a metal cylinder designed for irradiation of the entire rat’s body. A Lewis experimental rat is placed within a specially adapted metal cylinder designed for the procedure of whole-body irradiation. TBI, total body irradiation.

Fig 2.

Confirmation of the creation of chimeric cells via ex vivo PEG-mediated cell fusion. (a) The study design of the creation process of DRCC from isolated BM cells of ACI and Lewis rat donors. (b) Representative FC PKH26 vs. PKH67 fluorescent labeling dot plots of (from left): PKH26-stained ACI rat BM cells; PKH67-stained Lewis rat BM cells; and fused DRCC, double-stained with PKH26/PKH67 fluorescent dyes. (c) Representative immunofluorescence CM images of (from left): PKH26-stained ACI rat BM cells (red); PKH67-stained Lewis rat BM cells (green); and fused DRCC (orange color), revealing the overlapping PKH26/PKH67 fluorescent dyes, confirming the chimeric state of the created DRCC. Images were captured using an upright confocal microscope (Leica TCS-SP2, RRID:SCR_020231), original magnification: 400×, scale bar: 10 μm. BM, bone marrow; CM, confocal microscopy; DRCC, donor-recipient chimeric cell; FC, flow cytometry; PEG, polyethylene glycol.
Confirmation of the creation of chimeric cells via ex vivo PEG-mediated cell fusion. (a) The study design of the creation process of DRCC from isolated BM cells of ACI and Lewis rat donors. (b) Representative FC PKH26 vs. PKH67 fluorescent labeling dot plots of (from left): PKH26-stained ACI rat BM cells; PKH67-stained Lewis rat BM cells; and fused DRCC, double-stained with PKH26/PKH67 fluorescent dyes. (c) Representative immunofluorescence CM images of (from left): PKH26-stained ACI rat BM cells (red); PKH67-stained Lewis rat BM cells (green); and fused DRCC (orange color), revealing the overlapping PKH26/PKH67 fluorescent dyes, confirming the chimeric state of the created DRCC. Images were captured using an upright confocal microscope (Leica TCS-SP2, RRID:SCR_020231), original magnification: 400×, scale bar: 10 μm. BM, bone marrow; CM, confocal microscopy; DRCC, donor-recipient chimeric cell; FC, flow cytometry; PEG, polyethylene glycol.

Fig 3.

Survival rate changes post-7 Gy TBI with different cellular therapies over a 90-day observation. The highest mortality rate of 60% was observed following administration of the alloBMT therapy. In contrast, 100% survival rate was observed in all the remaining experimental groups. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; TBI, total body irradiation.
Survival rate changes post-7 Gy TBI with different cellular therapies over a 90-day observation. The highest mortality rate of 60% was observed following administration of the alloBMT therapy. In contrast, 100% survival rate was observed in all the remaining experimental groups. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; TBI, total body irradiation.

Fig 4.

Body weight changes post-7 Gy TBI with different cellular therapies during a 90-day observation. There were differences in the initial body mass between the animals assigned to the four experimental groups. The initial weight on day 0 was set as a baseline, denoted as 100%, and the subsequent weights were then calculated as the percentage relative to this baseline value. These percentage values were calculated in 10-day intervals until the end of the 90-day observation period. Ultimately, rats in all experimental groups regained their body weight, surpassing the initial values by the end of the observation period. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; TBI, total body irradiation.
Body weight changes post-7 Gy TBI with different cellular therapies during a 90-day observation. There were differences in the initial body mass between the animals assigned to the four experimental groups. The initial weight on day 0 was set as a baseline, denoted as 100%, and the subsequent weights were then calculated as the percentage relative to this baseline value. These percentage values were calculated in 10-day intervals until the end of the 90-day observation period. Ultimately, rats in all experimental groups regained their body weight, surpassing the initial values by the end of the observation period. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; TBI, total body irradiation.

Fig 5.

Cellular therapy’s impact on peripheral blood cell populations during 90-day observation post 7 Gy TBI. Assessment of the peripheral blood cells population counts after application of alloBMT, DRCC, and alloBMT + DRCC therapies following 7 Gy TBI confirmed the restoration of hematopoietic cells by DRCC therapy. The highest (a) WBC values were observed in the DRCC and alloBMT + DRCC groups throughout the entire 90 days follow-up. Also, the highest (b) lymphocyte values were observed in the DRCC and alloBMT + DRCC groups over the entire observation period. The only significant difference was observed in the (c) baseline monocyte values between the alloBMT + DRCC group and the DRCC therapy. No statistically significant differences were found in the values of (d) neutrophils or (e) eosinophils between all the experimental groups. The only difference in (f) basophil counts was observed in the baseline values of the alloBMT + DRCC and control groups. (g) RBC counts were comparable among the tested therapies, following a similar trend during the 90-day observation period. After the initial drop, (h) PLT counts exhibited an increase, with the highest values observed in the DRCC therapy group over the entire follow-up period. Data presented as mean ± SEM. One-way ANOVA test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. alloBMT, allogeneic bone marrow transplantation; ANOVA, analysis of variance; DRCC, donor–recipient chimeric cell; PLT, platelets; RBC, red blood cells; SEM, standard error of the mean; TBI, total body irradiation; WBC, white blood cells.
Cellular therapy’s impact on peripheral blood cell populations during 90-day observation post 7 Gy TBI. Assessment of the peripheral blood cells population counts after application of alloBMT, DRCC, and alloBMT + DRCC therapies following 7 Gy TBI confirmed the restoration of hematopoietic cells by DRCC therapy. The highest (a) WBC values were observed in the DRCC and alloBMT + DRCC groups throughout the entire 90 days follow-up. Also, the highest (b) lymphocyte values were observed in the DRCC and alloBMT + DRCC groups over the entire observation period. The only significant difference was observed in the (c) baseline monocyte values between the alloBMT + DRCC group and the DRCC therapy. No statistically significant differences were found in the values of (d) neutrophils or (e) eosinophils between all the experimental groups. The only difference in (f) basophil counts was observed in the baseline values of the alloBMT + DRCC and control groups. (g) RBC counts were comparable among the tested therapies, following a similar trend during the 90-day observation period. After the initial drop, (h) PLT counts exhibited an increase, with the highest values observed in the DRCC therapy group over the entire follow-up period. Data presented as mean ± SEM. One-way ANOVA test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. alloBMT, allogeneic bone marrow transplantation; ANOVA, analysis of variance; DRCC, donor–recipient chimeric cell; PLT, platelets; RBC, red blood cells; SEM, standard error of the mean; TBI, total body irradiation; WBC, white blood cells.

Fig 6.

Cellular therapy’s effect on kidney, skin, and small intestine histopathology 90 days post-7 Gy TBI. (a) Representative images of Hematoxylin and Eosin stained cross-sections of kidney, skin, and small intestine of Lewis rats at 90 days following 7 Gy TBI and application of: saline control, alloBMT, DRCC, and alloBMT + DRCC therapies. In renal tissue images, inflammatory changes and necrosis within the tubules were observed following alloBMT and alloBMT + DRCC therapies, respectively, as indicated by the arrow. Following DRCC therapy, no inflammation was observed within the examined tissues. Skin tissue images showed inflammatory infiltration of the appendages after alloBMT therapy, as demonstrated by the arrow. In the small intestine tissue sample, an apoptotic body marked by the arrow was noticed following alloBMT therapy. Magnification: 20 × . (b) The lowest incidence of GvHD on the renal tissue samples was observed in the DRCC group, compared to the highest incidence seen in the saline control group. Based on the assessment of the skin tissue samples, the highest score suggesting GvHD was recorded in the alloBMT group, whereas the lowest score was found in the DRCC group. Furthermore, the highest score of GvHD on slides of the small intestine samples was assessed in both the alloBMT and alloBMT + DRCC groups, while the lowest score was observed in the control group. Data presented as mean ± SEM. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; GvHD, graft-versus-host disease; SEM, standard error of the mean; TBI, total body irradiation.
Cellular therapy’s effect on kidney, skin, and small intestine histopathology 90 days post-7 Gy TBI. (a) Representative images of Hematoxylin and Eosin stained cross-sections of kidney, skin, and small intestine of Lewis rats at 90 days following 7 Gy TBI and application of: saline control, alloBMT, DRCC, and alloBMT + DRCC therapies. In renal tissue images, inflammatory changes and necrosis within the tubules were observed following alloBMT and alloBMT + DRCC therapies, respectively, as indicated by the arrow. Following DRCC therapy, no inflammation was observed within the examined tissues. Skin tissue images showed inflammatory infiltration of the appendages after alloBMT therapy, as demonstrated by the arrow. In the small intestine tissue sample, an apoptotic body marked by the arrow was noticed following alloBMT therapy. Magnification: 20 × . (b) The lowest incidence of GvHD on the renal tissue samples was observed in the DRCC group, compared to the highest incidence seen in the saline control group. Based on the assessment of the skin tissue samples, the highest score suggesting GvHD was recorded in the alloBMT group, whereas the lowest score was found in the DRCC group. Furthermore, the highest score of GvHD on slides of the small intestine samples was assessed in both the alloBMT and alloBMT + DRCC groups, while the lowest score was observed in the control group. Data presented as mean ± SEM. alloBMT, allogeneic bone marrow transplantation; DRCC, donor recipient chimeric cells; GvHD, graft-versus-host disease; SEM, standard error of the mean; TBI, total body irradiation.

Summary of experimental groups

Experimental group number Type of therapy applied Route of cells delivery Dosage of saline/number of cells injected Number of rats per group
Group 1 Saline Intraosseous 0.1 mL of saline n = 5
Group 2 alloBMT Intraosseous 80 × 106 cells n = 5
Group 3 DRCC Intraosseous 4–6 × 106 cells n = 5
Group 4 alloBMT + DRCC Intraosseous 80 × 106 cells + 4–6 × 106 cells n = 5
Total number of rats (n = 20)

Histopathological changes constituting the Small Criteria for assessing the degree of GvHD occurrence

Kidney Skin Small intestine
Tubulointerstitial inflammation, basement membrane nephropathy, microangiopathy, fibrinoid necrosis (segmental or global), interstitial fibrosis, tubular necrosis Vacuolar degeneration, epidermal necrosis, lichenification, inflammation of the skin appendages, non-specific lymphocytic dermatitis, apoptosis and dermal keratosis, fibrosis of the dermis, hyperkeratosis of the epidermis, acanthosis, spongiotic dermatitis with marked spongiosis Non-specific mucositis, gland damage, mucosal damage, the presence of crypt distortion, active inflammation, Paneth cell metaplasia, degree of mucosal fibrosis, apoptotic changes within crypts, the presence of intestinal endothelial lymphocytes

Criteria for assessing the degree of GvHD occurrence based on histopathological changes

Degree of GvHD occurrence (score) No GvHD (0) GvHD possible (1) Consistent with GvHD (2) Definite GvHD (3)
Kidney No deviation Presence of <2 small criteria Presence of ≥2 small criteria without the presence of basement membrane nephropathy Presence of basement membrane nephropathy
Skin Presence of one small criterion or the absence of deviations Presence of ≥2 small criteria or the presence of apoptosis Presence of lichenification, dermal fibrosis, spongiosis without apoptosis, or presence of apoptosis with small criteria but excluding lichenification and spongiosis Occurrence of apoptosis with lichenification and acanthosis or spongiosis
Small intestine Presence of one small criterion or the absence of deviations Presence of ≥2 small criteria or small criteria without the presence of crypt apoptosis Presence of ≥2 small criteria in the presence of 2–5 apoptotic crypts Presence of >6 apoptotic crypts with at least one small criterion

Comparison of clinical parameters after 7 Gy TBI and different cellular therapy administration

Experimental groups Activity in the cage (0–3) Body posture (0–3) Fur coverage (0–3) Overall physical condition total score (0–9) Blood in stool (0–1) Diarrhea incidence (0–1) Digestive distress condition total score (0–2)
Saline control 0.96 ± 0.453* 0.02 ± 0.026 0.70 ± 0.089 1.68 0.18 ± 0.187 0.16 ± 0.118* 0.34
alloBMT 0.13 ± 0.031* 0.34 ± 0.227* 1.15 ± 0.273* 1.62 0.19 ± 0.194* 0.09 ± 0.025 0.28
DRCC 0.25 ± 0.303 0.02 ± 0.027 0.51 ± 0.118* 0.78 0.06 ± 0.025 0.00 ± 0.009* 0.06
alloBMT + DRCC 0.29 ± 0.302 0.00 ± 0.000* 0.60 ± 0.094 0.89 0.07 ± 0.023* 0.08 ± 0.027 0.15

Clinical parameters evaluated over 90 days post-7 Gy TBI and different cellular therapy administrations

Clinical parameters Ratings Score system
Activity in the cage

0- Very active animal, moving in the cage, typical gait

1- Slightly decreased degree of activity, gait mildly disturbed

2- Animal moving very slowly, gait severely disturbed

3- Animal reluctant to move or lack of any movement

 		0–3 points
Body posture

0- Normal body posture

1- Slightly hunched over

2- Moderately hunched over

3- Very hunched over

 		0–3 points
Fur coverage

0- Normal fur condition

1- No fur loss and spiky fur

2- Moderate fur loss and raised fur

3- Significant fur loss and bristling fur

 0–3 points
Overall physical condition total score = 0–9 points
Blood in stool incidence

0- Absence of blood in stool

1- Presence of blood in stool

 		0–1 point
Diarrhea incidence

0- Absence of diarrhea, normal stool consistency

1- Presence of diarrhea

 		0–1 point
Digestive distress condition total score = 0–2 points
eISSN:
1661-4917
Lingua:
Inglese
Frequenza di pubblicazione:
Volume Open
Argomenti della rivista:
Medicine, Basic Medical Science, Biochemistry, Immunology, Clinical Medicine, other, Clinical Chemistry
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