The chick embryo is a favorite and great in vivo model, which has long been used in embryology, developmental biology and applied sciences. Also, chicken embryo is the accessible model for transplantation and tracking studies. Avian multipotent stem cells have the capability to self-renew and differentiate into a variety of cell types in vitro. Avian multipotent stem cells were successfully isolated from bone marrow (BM), metanephric tissue, lung, amniotic cells, cartilage, and other embryonic and adult tissues. Subsequent analysis was performed to evaluate these multipotent cells in vitro. Results of the present study strongly contribute to the information related to the ability of avian multipotent stem cells to differentiate into cells such as adipocytes, osteoblasts, chondrocytes, astrocytes, and other committed cells. Avian multipotent stem cells may provide a new source of avian-derived cell lines for basic research and potential therapeutic applications.

Keywords: avian stem cells, multipotent stem cells, mesenchymal stem cells, regenerative medicine

One of the first most important events in the avian embryonic development is the specification of the three embryonic lineage including ectoderm, mesoderm, and endoderm.^1,2 The commitment of pluripotent embryonic cells into these groups requires the continue action of several gene products. Although, in the adult tissue, the presence of multipotent to unipotent cells have been considered to maintain some tissue regeneration.^3‒7 Avian embryos are a great model in basic and applied sciences.^8,9 Avian multipotent stem cells can maintain self-renewal and multilineage differentiation ability in vitro.¹⁰ The vast majority of avian multipotent stem cells have been obtained from different tissue sources including bone marrow, primary kidney, lung, dermis, and other tissues and the stem cell populations derived from these tissues are valuable (Figure 1). Among different kinds of multipotent stem cells, mesenchymal stem cells are the most representative type, which can be differentiate into muscle, adipocytes, cartilage, and osteoblasts in vitro.¹¹ However, the fate of multipotent stem cells was believed to differentiate only into the tissue or organ from which they originated.^6,12 Although, previous reports have suggested that in specific environmental conditions, the multipotent cells may undergo unexpected fate.^6,12 The appropriate effects of multipotent stem cells are in cell-based therapy, tissue engineering, cell transplantation, transgenic studies, and viral vaccines production.^9,13‒16 Therefore, these cells may provide a new source of avian-derived cell lines for basic research and potential therapeutic applications.^17‒19

Figure 1 Avian multipotent stem cells. Morphological properties of avian multipotent stem cells. The appropriate effects of these cells are in cell-based therapy, tissue engineering, cell transplantation, and transgenic studies, (MSCs; mesenchymal stem cells).

Avian mesenchymal stem cells

Avian mesenchymal stem cells (MSCs) are a small population and commonly isolated from adult bone marrow (BM).^10,12,20,21 MSCs are fibroblast-like multipotent progenitor cells which have the ability for self-renewal and a differentiation capacity.²² MSCs were isolated from different anatomical regions of chicken embryo²³ and postnatal 1-14days old chicken.^11,20 MSCs have been isolated from lung,²² adipose tissue, and skeletal muscle, and metanephric tissue, as well.¹⁴ These cells are positive for classic embryonic stem cells that are associated with self-renewal (Table 1)^20,24 and differentiation capacity into muscle, adipocytes, cartilage, and osteoblasts in vitro (Figure 2).^6,24,25 Moreover, they have immunoregulatory function and inhibit immunological response,^26,27 therefore, these cells are permissive to infected with a variety of pathogenic viruses such as infectious bursal disease virus (IBDV)¹³ which does not proliferate in chicken fibroblast cells.²⁴ MSCs are a great tool for a wide variety of diseases research such as cell transplantation, regenerative medicine, and production of transgenic chickens.^12,28

Figure 2 Potential of multi-lineage avian mesenchymal stem cells (MSCs). MSCs are fibroblast-like multipotent progenitor cells which commonly isolated from adult bone marrow (BM) and have the ability for self-renewal and a differentiation capacity to muscle cells, adipocytes, cartilage, and osteoblasts in vitro.

Avian metanephric mesenchymal stem cells

Avian metanephric mesenchymal stem cells (MMSCs) was obtained from 20days old Beijing duck metanephros.¹⁴ The expression of related genes and proteins, self-renewal potential, their differentiation, and similar morphology with MSCs were evaluated.^14,29 However, their specific surface markers are unknown and they identified based on the expression of certain markers associated with MSCs (Table 1). It seems that, MMSCs are pluripotent and able to differentiate into three somatic lineages such as epithelial cells, liver cells, and pancreatic cells.¹⁴ There are few reports of harvesting avian MMSCs; while, MMSCs are applicable for tissue engineering, cell transplantation, and cell therapy applications on the kidney.^14,30

Avian lung mesenchymal stem cells

Avian Lungs derived mesenchymal stem cells (LMSCs) were obtained from 1 to 2 weeks old chicken. Chicken LMSCs similar to human³¹ and mouse LMSCs³² are multipotent cells with a fibroblast-like morphology, expression of self-renewal and pluripotency marker (Table 1) and differentiation into adipocytes and osteocytes.²² LMSCs have immuneregulatory properties by the inhibition of T-cell proliferation; indeed they have immunosuppressive effects. LMSCs are susceptible to infection with pathogenic viruses and suitable target for viral replication and apoptosis. LMSCs are valuable source for cell therapy applications on the lung and as target for virus infection.²²

Avian amniotic mesenchymal stem cells

Avian amniotic mesenchymal stem cells (AMSCs) were obtained from amniotic epithelial cells.³³ AMSCs are multipotent cells and express transcription factors which are essential for maintaining the self-renewal and undifferentiated state (Table 1). AMSCs are able to give rise into osteoblasts, adipocytes, myocardial cells,³⁴ neural-like cells, and islet like cells.³³ AMSCs suitable tool for tissue engineering research and autologous cell therapy.^34‒36

Avian cartilage stem/progenitor cells

Avian cartilage-derived stem/progenitor cells (CSPCs) were obtained from the surface zone of articular cartilage from 20days old chicken embryo. CSPCs are uniform population with long fusiform or polygon morphology.³⁷ CSPCs are multipotent stem cells and identify by expression of specific surface markers, ability to self-renew, and differentiate into three cell types such as adipocytes, osteoblasts, and chondrocytes.^37,38 These cells are great tools for cell-based cartilage repair.^37,39-41

Avian neural stem/progenitor cells

Avian neural stem and progenitor cells (NSPCs) were isolated from dorsal ventricular ridges of 10-13days old chick embryos.⁴² NSPCs are round neurospheres which able to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes.⁴² These cells are suitable for gene therapy, intracellular distribution of proteins, neurogenesis, tumorigenesis, and given rise to many interesting findings.^42‒45

Avian dermis-derived mesenchymal stem/progenitor cells

Avian dermis-derived mesenchymal stem/progenitor cells (DMS/PCs) were obtained from dermis of 16days old chick embryos.⁴⁶ These cells show fibroblast-like morphology and multilineage differentiation into adipocyte, osteocytes, and neural cells.⁴⁶ DMS/PCs exhibit extensive applications in stem-cell based therapy and tissue regeneration study.^46‒48

Avian adipose-derived stem/progenitor cells

Avian adipose-derived stem/progenitor cells (ADPCs) were isolated from subcutaneous tissues of the abdomen and inguinal fat pads of 1 day old chickens.⁴⁹ ADPCs are fibroblast-like multipotent stem cells with self-renewal ability and differentiation into osteoblasts, adipocytes, and myocardial cells in vitro.^17,49 These cells are novel tool for regenerative medicine, as well.^17,49‒53

The biochemical and molecular markers for detecting and evaluating avian multipotent stem cells have yet to be developed (Table 1). In vitro studies have also demonstrated the advantages of using multipotent stem cells for cell transplantation. Actually, very few researches exist that incorporate the beneficial effects of avian multipotent stem cells. Collectively, these data demonstrate that research on the properties of avian multipotent stem cells have led to renewed interest in their extended applications.

This study was financially supported by grants provided from Royan Institute and Iranian Council of Stem Cell Technology and the Iran National Science Foundation (INSF).

Author declares that there is no conflict of interest.

1. Starck JM. Avian growth and development: evolution within the altricial-precocial spectrum. USA: Oxford University Press; 1998.
2. Painter KJ, Maini PK, Othmer HG. A chemotactic model for the advance and retreat of the primitive streak in avian development. Bull Math Biol. 2000;62(3):501‒525.
3. Raff M. Adult stem cell plasticity: fact or artifact? Annual review of cell and developmental biology. 2003;19(1):1‒22.
4. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med. 2004;8(3):301‒316.
5. Eisenberg LM, Eisenberg CA. Stem cell plasticity, cell fusion, trans differentiation. Birth Defects Research Part C: Embryo Today: Reviews. 2003;69(3):209‒218.
6. Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell. 2004;116(5):639‒648.
7. Clevers H. What is an adult stem cell? Science. 2015;350(6266):1319‒1320.
8. Farzaneh M, Khoshnam SE, Nokhbatolfoghahai M. First scientific record of two cases of partial twinning in the chick embryo, Gallus gallus domesticus. Veterinary Record Case Reports. 2016;4(2):e000353.
9. Mozdziak PE, Petitte JN. Status of transgenic chicken models for developmental biology. Dev Dyn. 2004;229(3):414‒421.
10. Bai C, Hou L, Ma Y, et al. Isolation and characterization of mesenchymal stem cells from chicken bone marrow. Cell Tissue Bank. 2013;14(3):437‒451.
11. Bhuvanalakshmi G, Arfuso F, Dharmarajan A, et al. Multifunctional properties of chicken embryonic prenatal mesenchymal stem cells-pluripotency, plasticity, and tumor suppression. Stem Cell Rev. 2014;10(6):856‒870.
12. Young Tae Heo, Sung Ho Lee, Ji Hoon Yang, et al. Bone marrow cell-mediated production of transgenic chickens. Laboratory Investigation. 2011;91(8):1229‒1240.
13. Khatri M, Sharma JM. Susceptibility of chicken mesenchymal stem cells to infectious bursal disease virus. Journal of virological methods. 2009;160(1):197‒199.
14. Chen J, Pu Y, Sun Y, et al. Biological characterization of metanephric mesenchymal stem cells from the Beijing duck. Experimental and therapeutic medicine. 2016;11(2):439‒447.
15. Petitte JN, PE Mozdziak. Production of transgenic poultry. Transgenic technology: a laboratory handbook. 2nd ed. New York: Elsevier Science; 2002. p. 279‒306.
16. Tingyu Dai, Liao Wu, Zelin Chen, et al. Stem Cell Based Biotherapy for Radiation Related Injury. Advanced Trauma and Surgery. 2017. p. 357‒385.
17. Mizuno H, Tobita M, Uysal AC. Concise review: adipose‐derived stem cells as a novel tool for future regenerative medicine. Stem cells. 2012;30(5):804‒810.
18. Rachel A Stern, Srinivasan Dasarathy, Paul E Mozdziak. Ammonia elicits a different myogenic response in avian and murine myotubes. In Vitro Cellular & Developmental Biology-Animal. 2016:1‒12.
19. Nierobisz LS, McFarland DC, Mozdziak PE. MitoQ 10 induces adipogenesis and oxidative metabolism in myotube cultures. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2011;158(2):125‒131.
20. Khatri M, O'Brien TD, Sharma JM. Isolation and differentiation of chicken mesenchymal stem cells from bone marrow. Stem Cells Dev. 2009;18(10):1485‒1492.
21. Li L, Bai X, Gong X, et al. Differentiation potential of bone marrow mesenchymal stem cells in duck. J Genet Genomics. 2009;36(3):133‒140.
22. Khatri M, O'Brien TD, Goyal SM, et al. Isolation and characterization of chicken lung mesenchymal stromal cells and their susceptibility to avian influenza virus. Dev Comp Immunol. 2010;34(4):474‒479.
23. Young HE, Ceballos EM, Smith JC, et al. Pluripotent mesenchymal stem cells reside within avian connective tissue matrices. In Vitro Cell Dev Biol Anim. 1993;29(9):723‒736.
24. Calloni R, Viegas GS, Türck P, et al. Mesenchymal stromal cells from unconventional model organisms. Cytotherapy. 2014;16(1):3‒16.
25. Martin I, Padera RF, Vunjak-Novakovic G, et al. In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone‐like tissues. J Orthop Res. 1998;16(2):181‒189.
26. Iyer SS, Rojas M. Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies. Expert Opin Biol Ther. 2008;8(5):569‒581.
27. Fierabracci A, Del Fattore A, Muraca M. The Immunoregulatory Activity of Mesenchymal Stem Cells: 'State of Art' and 'Future Avenues'. Curr Med Chem. 2016;23(27):3014‒3024.
28. Nitkin CR, Bonfield TL. Concise Review: Mesenchymal Stem Cell Therapy for Pediatric Disease: Perspectives on Success and Potential Improvements. Stem Cells Transl Med. 2015;6(2):539‒565.
29. Duffield JS, Park KM, Hsiao LL, et al. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. J Clin Invest. 2005;115(7):1743‒1755.
30. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol. 2000;28(8):875‒884.
31. Lama VN, Smith L, Badri L, Flint A, Andrei AC, et al. (2007) Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts. J Clin Invest. 2007;117(4):989‒996.
32. Summer R, Fitzsimmons K, Dwyer D, et al. Isolation of an adult mouse lung mesenchymal progenitor cell population. Am J Respir Cell Mol Biol. 2007;37(2):152‒159.
33. Gao Y, Pu Y, Wang D, et al. Isolation and biological characterization of chicken amnion epithelial cells. Eur J Histochem. 2012;56(3):e33.
34. Li X, Gao Y, Hua J, et al. Research potential of multi-lineage chicken amniotic mesenchymal stem cells. Biotech Histochem. 2014;89(3):172‒180.
35. Mamede AC, MF Botelho. Amniotic Membrane: Origin, Characterization and Medical Applications. Netherlands: Springer; 2015.
36. Feng C, D Graham C, Connors JP, et al. A comparison between placental and amniotic mesenchymal stem cells for transamniotic stem cell therapy (TRASCET) in experimental spina bifida. J Pediatr Surg. 2016;51(6):1010‒1013.
37. Li L, Ma Y, Li X, et al. Isolation, Culture, and Characterization of Chicken Cartilage Stem/Progenitor Cells. Biomed Res Int. 2015;2015:586290.
38. Karlsson C, Lindahl A. Articular cartilage stem cell signalling. Arthritis Res Ther. 2009;11(4):121.
39. Jayasuriya CT, Chen Y, Liu W, et al. The influence of tissue microenvironment on stem cell–based cartilage repair. Ann N Y Acad Sci. 2016;1383(1):21‒33.
40. Jiang Y, Tuan RS. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol. 2015;11(4):206‒212.
41. Shafiee A, Kabiri M, Langroudi L, et al. Evaluation and comparison of the in vitro characteristics and chondrogenic capacity of four adult stem/progenitor cells for cartilage cell‐based repair. J Biomed Mater Res A. 2016;104(3):600‒610.
42. Hou L, Jin D, Gu E, et al. Isolation and characterization of duck embryonic neural stem and progenitor cells. Poult Sci. 2011;90(3):609‒617.
43. Xu C, Loh HH, Law PY. Effects of addictive drugs on adult neural stem/progenitor cells. Cell Mol Life Sci. 2016;73(2):327‒348.
44. Covacu R, Brundin L. Effects of neuroinflammation on neural stem cells. Neuroscientist. 2015;1073858415616559.
45. Bengoa-Vergniory N, Kypta RM. Canonical and noncanonical Wnt signaling in neural stem/progenitor cells. Cell Mol Life Sci. 2015;72(21):4157‒4172.
46. Gao Y, Bai C, Xiong H, et al. Isolation and characterization of chicken dermis-derived mesenchymal stem/progenitor cells. Biomed Res Int. 2013;2013:626258.
47. Chen Z, Wang Y, Shi C. Therapeutic implications of newly identified stem cell populations from the skin dermis. Cell Transplant. 2015;24(8):1405‒1422.
48. Park JR, Kim E, Yang J, et al. Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization. Cell Tissue Bank. 2015;16(2):209‒218.
49. Gong X, Hou L, Bai C, et al. Isolation and biological characteristics of chicken adipose-derived progenitor cells. DNA Cell Biol. 2011;30(7):453‒460.
50. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249‒1260.
51. Mizuno H, Morikuni Tobita, Hakan Orbay A, et al. Adipose-Derived Stem Cells as a Novel Tool for Future Regenerative Medicine. Stem Cells and Cancer Stem Cells Springer. 2014. p. 165‒174.
52. Kapur SK, Dos-Anjos Vilaboa S, Llull R, et al. Adipose tissue and stem/progenitor cells: discovery and development. Clin Plast Surg. 2015;42(2): 155‒167.
53. Fiona A van Vollenstee, Carla Dessels, Karlien Kallmeyer, et al. Isolation and Characterization of Adipose-Derived Stromal Cells. Stem Cell Processing Springer. 2016. p. 131‒161.