Int J Stem Cells.  2016 May;9(1):96-106. 10.15283/ijsc.2016.9.1.96.

Therapeutic Potential of Bone Marrow-Derived Mesenchymal Stem Cells on Experimental Liver Injury Induced by Schistosoma mansoni: A Histological Study

• Fikry H
• Abdel Gawad S
• Baher W

• KMID: 2164166
• DOI: http://doi.org/10.15283/ijsc.2016.9.1.96

Abstract

BACKGROUND AND OBJECTIVES
Bone marrow derived mesenchymal stem cells (BM-MSCs) have been proposed as effective treatment of many diseases owing to their unique ability to differentiate into other cell types in vivo. Schistosoma mansoni (S. mansoni) infection is characterized by hepatic granuloma formation around schistosome eggs at acute stage of infection, followed by hepatic fibrosis at chronic and advanced stages. Whether BM-MSCs have an ameliorative effect on hepatic tissue injury caused by S. mansoni infection or not, was inspected in the current study. MATERIALS AND RESULTS: Female Swiss Albino mice were divided into a control group and an experimental group. Half of control animals served as donors for bone marrow stem cells, and the other half was used to collect liver samples. Experimental group was injected with circariae of S. mansoni, and then subdivided into three subgroups; Subgroup B1, sacrificed after eight weeks of infection without treatment, subgroup B2, received BM-MSCs at the eighth week and sacrificed four weeks later, and subgroup B3, was untreated till the twelfth week of infection. Histological examination of liver samples showed the formation of granulomas and liver fibrosis which were extensive in subgroup B3. However, treated subgroup illustrated improvement of liver histology, signs of hepatocytes regeneration, and possible contribution of oval cell in the process of hepatic and biliary regeneration.
CONCLUSION
BM-MSCs decreased liver fibrosis and contributed to an increase in oval cells, generation of new hepatocytes and/or to the improvement of resident hepatocytes in S. mansoni infected mice.

Keyword

S. mansoni; Mesenchymal stem cells; Liver fibrosis; Oval cells

MeSH Terms

Animals
Bone Marrow
Eggs
Female
Fibrosis
Granuloma
Hepatocytes
Humans
Liver Cirrhosis
Liver*
Mesenchymal Stromal Cells*
Mice
Ovum
Regeneration
Schistosoma mansoni*
Schistosoma*
Stem Cells
Tissue Donors

Figure

• 

  Fig. 1 (A) Culture on day five, showing adherent fibroblast like cells with multiple processes. They have granular cytoplasm and vesicular nuclei. Some nuclei appear with two nucleoli (↑). The non-adherent cells appear rounded and refractile (Δ). (B) Culture on day nine, showing confluent dense homogenous fibroblast like cells with granular cytoplasm and multiple interdigitating process (↑). Notice the presence of few rounded and refractile non-adherent cells (Δ). (C) Streptavidin-biotin peroxidase staining for CD44 showing that most of the cultured cells exhibit positive brownish reaction for CD44 (↑). (D) BM-MSCs incubated in osteogenic media for three weeks stained with Alizarin red. Red staining (↑) demonstrates mineralised matrix produced by osteoblastic differentiation (phase contrast microscope X200).

• 

  Fig. 2 (A) Show classical hepatic lobules with plates of hepatocytes radiating from the central vein (cv) separated by blood sinusoids (↑) (Control, H&E ×160). (B) Show many scattered bilharizial granulomas around S. mansoni eggs (circumscribed areas) (Subgroup B1, H&E ×160). (C) Show small few bilharizial granulomas (circumscribed areas) with normal hepatic tissue inbetween (*) (Subgroup B2, H&E ×160). (D) Show many large bilharizial granulomas (circumscribed areas) occupying most of hepatic tissue sparing only few areas of hepatocytes (*) (Subgroup B3, H&E ×160).

• 

  Fig. 3 (A) Show cells with small oval shaped centrally located nuclei along the periphery of the granulomatous lesions (↓) (Subgroup B2, H&E ×640). (B) Small oval shaped cells are absent around granulomatous lesions (Subgroup B3, H&E ×640). (C) Show proliferating hepatic duct lined with columnar cells having basal nuclei (↓) (Subgroup B2, H&E ×640). (D) Show bile duct with disintegrated and highly vacuolated epithelium (↓) (Subgroup B3, H&E ×640).

• 

  Fig. 4 (A) Show few collagen fibers around central veins and portal areas (↑) (Control, Masson’s trichrome ×160). (B) Collagen fibers are seen in granulomatous lesions (↑) (Subgroup B1, Masson’s trichrome ×160). (C) Few collagen fibers are present in granulomatous lesions (↑) sparing most of the hepatic tissue (Subgroup B2, Masson’s trichrome ×160). (D) Abundant collagen fibers occupy granulomatous lesions (↑) and form bridging fibrosis inbetween them (<), sparing only few areas of hepatic tissue (*) (Subgroup B3, Masson’s trichrome x160).

• 

  Fig. 5 (A) Show few CK7 positive cells lining and around a bile duct (↑) (Control, immunostaining for CK7 ×640). (B) Very few CK7 positive cells along the periphery of granulomatous lesions (↑) (Subgroup B1, immunostaining for CK7 ×640). (C) Many CK7 positive cells are seen along the periphery of granulomatous lesions (↑) (Subgroup B2, immunostaining for CK7 ×640). (D) Few CK7 positive cells are seen near a granulomatous lesion (↑) (Subgroup B3, immunostaining for CK7 ×640).

• 

  Fig. 6 (A) PKH26- labeled MSCs are seen scattered in the liver tissue of treated group (fluorescent microscope, Subgroup B2). (B) Show a hepatocyte with large rounded, central vesicular nucleus (N). The nucleus shows the usual characteristic chromatin distribution. The cytoplasm contains numerous mitochondria (M), rER (↑), and glycogen rosettes (*) (Control, TEM ×10000). (C) Show part of a hepatocyte with few microvilli projecting from the surface facing the space of Disse (↑), HSCs (*) are seen in the perisinusoidal space with many fat droplets (F) in the cytoplasm. A bile canaliculus appears in the form of its microvilli (<) (Control, TEM ×10000). (D) Show an increase of collagen fibers (*) in between necrotic hepatocytes that have abnormal distribution of nuclear chromatin (↑), damage of a bile canaliculus in the form of partial loss of its microvilli (thick ↑). Eosinophil cells (E) appear in the blood sinusoids with their specific granules (Subgroup B1, TEM ×8000).

• 

  Fig. 7 (A) Show a large premature hepatocyte with signs of regeneration in the form of irregular nuclear membrane (thick arrow) and an apparent increase of organelles e.g. mitochondria (M), rER (↑) and free ribosomes (R) (subgroup B2, TEM ×10000). (B) Show part of hepatocyte with few microvilli projecting from the surface facing the blood sinusoid (↑). A bile canaliculus appears in the form of its microvilli (thick ↑) nearly as the control group (Subgroup B2, TEM X10000). (C) Show damage to a bile canaliculus in the form of severe loss of its microvilli (↑). An apparent increase of collagen fibers appear in between necrotic hepatocytes (*) (Subgroup B3, TEM ×10000). (D) Show damage to a bile canaliculus in the form of partial loss of its microvilli (↑). HSCs (*) are seen in the perisinusoidal space (Subgroup B3, TEM ×10000).

Reference

References

1. Patz JA, Graczyk TK, Geller N, Vittor AY. Effects of environmental change on emerging parasitic diseases. Int J Parasitol. 2000; 30:1395–1405. DOI: 10.1016/S0020-7519(00)00141-7. PMID: 11113264.

2. Oliveira SA, Souza BS, Guimaraes-Ferreira CA, Barreto ES, Souza SC, Freitas LA, Ribeiro-Dos-Santos R, Soares MB. Therapy with bone marrow cells reduces liver alterations in mice chronically infected by Schistosoma mansoni. World J Gastroenterol. 2008; 14:5842–5850. DOI: 10.3748/wjg.14.5842. PMID: 18855983. PMCID: 2751894.

3. Xiao SH, Mei JY, Jiao PY. Schistosoma japonicum-infected hamsters (Mesocricetus auratus) used as a model in experimental chemotherapy with praziquantel, artemether, and OZ compounds. Parasitol Res. 2011; 108:431–437. DOI: 10.1007/s00436-010-2084-7.

4. Ross AG, Bartley PB, Sleigh AC, Olds GR, Li Y, Williams GM, McManus DP. Schistosomiasis. N Engl J Med. 2002; 346:1212–1220. DOI: 10.1056/NEJMra012396. PMID: 11961151.

5. Xu H, Qian H, Zhu W, Zhang X, Yan Y, Mao F, Wang M, Xu H, Xu W. Mesenchymal stem cells relieve fibrosis of Schistosoma japonicum-induced mouse liver injury. Exp Biol Med (Maywood). 2012; 237:585–592. DOI: 10.1258/ebm.2012.011362.

6. Lorenzini S, Gitto S, Grandini E, Andreone P, Bernardi M. Stem cells for end stage liver disease: how far have we got? World J Gastroenterol. 2008; 14:4593–4599. DOI: 10.3748/wjg.14.4593. PMID: 18698672. PMCID: 2738783.

7. Salama H, Zekri AR, Bahnassy AA, Medhat E, Halim HA, Ahmed OS, Mohamed G, Al Alim SA, Sherif GM. Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease. World J Gastroenterol. 2010; 16:5297–5305. DOI: 10.3748/wjg.v16.i42.5297. PMID: 21072892. PMCID: 2980678.

8. Ishii K, Yoshida Y, Akechi Y, Sakabe T, Nishio R, Ikeda R, Terabayashi K, Matsumi Y, Gonda K, Okamoto H, Takubo K, Tajima F, Tsuchiya H, Hoshikawa Y, Kurimasa A, Umezawa A, Shiota G. Hepatic differentiation of human bone marrow-derived mesenchymal stem cells by tetracycline-regulated hepatocyte nuclear factor 3beta. Hepatology. 2008; 48:597–606. DOI: 10.1002/hep.22362. PMID: 18666263.

9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8:315–317. DOI: 10.1080/14653240600855905. PMID: 16923606.

10. Horan PK, Melnicoff MJ, Jensen BD, Slezak SE. Fluorescent cell labeling for in vivo and in vitro cell tracking. Methods Cell Biol. 1990; 33:469–490. DOI: 10.1016/S0091-679X(08)60547-6. PMID: 2084480.

11. Boros DL. Immunopathology of Schistosoma mansoni infection. Clin Microbiol Rev. 1989; 2:250–269. PMID: 2504481. PMCID: 358119.

12. Baddamwar A. Hepatic fibrosis in experimental Schistosoma japonicum infection in pigs: a histological and immunohistochemical study. Swedish University of Agriculture Sciences;Uppsala, Sweden: 2004. Report no. 46. 1403-2201.

13. Ghanem LY, Dahmen U, Dirsch O, Nosseir MM, Mahmoud SS, Mansour WA. Does granulocyte-colony stimulating factor administration induce damage or repair response in schistosomiasis? World J Hepatol. 2010; 2:434–441. DOI: 10.4254/wjh.v2.i12.434. PMID: 21191519. PMCID: 3010513.

14. Herrera MB, Bussolati B, Bruno S, Morando L, Mauriello-Romanazzi G, Sanavio F, Stamenkovic I, Biancone L, Camussi G. Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury. Kidney Int. 2007; 72:430–441. DOI: 10.1038/sj.ki.5002334. PMID: 17507906.

15. Gressner AM, Weiskirchen R, Breitkopf K, Dooley S. Roles of TGF-beta in hepatic fibrosis. Front Biosci. 2002; 7:d793–d807. DOI: 10.2741/A812. PMID: 11897555.

16. Cho IJ, Kim YW, Han CY, Kim EH, Anderson RA, Lee YS, Lee CH, Hwang SJ, Kim SG. E-cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation. Hepatology. 2010; 52:2053–2064. DOI: 10.1002/hep.23931. PMID: 20890948. PMCID: 3086490.

17. Fang B, Shi M, Liao L, Yang S, Liu Y, Zhao RC. Systemic infusion of FLK1(+) mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice. Transplantation. 2004; 78:83–88. DOI: 10.1097/01.TP.0000128326.95294.14. PMID: 15257043.

18. Rygiel KA, Robertson H, Marshall HL, Pekalski M, Zhao L, Booth TA, Jones DE, Burt AD, Kirby JA. Epithelial-mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease. Lab Invest. 2008; 88:112–123. DOI: 10.1038/labinvest.3700704.

19. Pulavendran S, Vignesh J, Rose C. Differential anti-inflammatory and anti-fibrotic activity of transplanted mesenchymal vs. hematopoietic stem cells in carbon tetrachloride-induced liver injury in mice. Int Immunopharmacol. 2010; 10:513–519. DOI: 10.1016/j.intimp.2010.01.014. PMID: 20144743.

20. El-Mahdi MM, Mansour WA, Hammam O, Mehana NA, Hussein TM. Ameliorative effect of bone marrow-derived stem cells on injured liver of mice infected with Schistosoma mansoni. Korean J Parasitol. 2014; 52:151–162. DOI: 10.3347/kjp.2014.52.2.151. PMID: 24850958. PMCID: 4028452.

21. Kordes C, Häussinger D. Hepatic stem cell niches. J Clin Invest. 2013; 123:1874–1880. DOI: 10.1172/JCI66027. PMID: 23635785. PMCID: 3638908.

22. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science. 1999; 284:1168–1170. DOI: 10.1126/science.284.5417.1168. PMID: 10325227.

23. Lenzi R, Liu MH, Tarsetti F, Slott PA, Alpini G, Zhai WR, Paronetto F, Lenzen R, Tavoloni N. Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells. Lab Invest. 1992; 66:390–402. PMID: 1538592.

24. Song S, Witek RP, Lu Y, Choi YK, Zheng D, Jorgensen M, Li C, Flotte TR, Petersen BE. Ex vivo transduced liver progenitor cells as a platform for gene therapy in mice. Hepatology. 2004; 40:918–924. DOI: 10.1002/hep.1840400422. PMID: 15382177.

25. Theise ND, Saxena R, Portmann BC, Thung SN, Yee H, Chiriboga L, Kumar A, Crawford JM. The canals of Hering and hepatic stem cells in humans. Hepatology. 1999; 30:1425–1433. DOI: 10.1002/hep.510300614. PMID: 10573521.

26. Novikoff PM, Yam A, Oikawa I. Blast-like cell compartment in carcinogen-induced proliferating bile ductules. Am J Pathol. 1996; 148:1473–1492. PMID: 8623918. PMCID: 1861562.

27. Yovchev MI, Grozdanov PN, Joseph B, Gupta S, Dabeva MD. Novel hepatic progenitor cell surface markers in the adult rat liver. Hepatology. 2007; 45:139–149. DOI: 10.1002/hep.21448.

28. Vig P, Russo FP, Edwards RJ, Tadrous PJ, Wright NA, Thomas HC, Alison MR, Forbes SJ. The sources of parenchymal regeneration after chronic hepatocellular liver injury in mice. Hepatology. 2006; 43:316–324. DOI: 10.1002/hep.21018. PMID: 16440343.

29. Elkhafif N, Voss B, Hammam O, Yehia H, Mansy S, Akl M, Boehm S, Mahmoud S, El Bendary O, El Fandy G. Homing of transplanted bone marrow cells in livers of Schistosoma mansoni-infected mice. APMIS. 2010; 118:277–287. DOI: 10.1111/j.1600-0463.2010.02585.x. PMID: 20402673.