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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 70  |  Issue : 3  |  Page : 136-139

Proliferative capacity of retinal progenitor cells in human fetal retina


1 Department of Anatomy, MGM Medical College, Navi Mumbai, Maharashtra, India
2 Department of Anatomy, MGM Medical College and Hospital, Navi Mumbai, Maharashtra, India

Date of Submission30-May-2021
Date of Acceptance23-Aug-2021
Date of Web Publication23-Sep-2021

Correspondence Address:
Dr. Anjali Satyen Sabnis
Department of Anatomy, MGM Medical College and Hospital, Kamothe, Navi Mumbai . 410 209, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jasi.jasi_100_21

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  Abstract 


Introduction: Retina is an innermost, delicate, and photosensitive layer of the eyeball, which is composed of 10 layers and 8 specialized cells which are involved in paramount function of the body like vision. Retinal neurogenesis commences from the layers of optic cup, which forms from optic vesicle. Progenitor cells are the tissue-specific cells which give rise to all different types of retinal cells. Progenitor cells in fetal retina proliferate at specific time during development of retina. Knowledge of the highest proliferative capacity interval of progenitor cells will be valuable for transplantation. Material and Methods: Twenty-eight fetuses of spontaneous abortions of 13th–40th week were collected from MGM Hospital after ethical and scientific approval of the institute. After fixation of fetuses, eyeballs were extracted and fixed in buffer solution. Sections were taken and the retina was treated with Ki-67 immunohistochemistry marker to observe proliferative capacity of retinal progenitor cells (RPCs). Seven groups (A to G) of 4 weeks were made and observations of each group were noted. Results: It was observed that the highest proliferative capacity of RPCs was in B group (17–20 weeks) and the highest proliferative capacity of RPCs was maximum at 19th week of gestation. Discussion and Conclusion: Characteristics of progenitor cells in retina are well studied. Their highest proliferation period can be utilized to make the procedure of transplantation more refined.

Keywords: Fetal retina, progenitor cells, proliferation, retina


How to cite this article:
Mane P, Sabnis AS. Proliferative capacity of retinal progenitor cells in human fetal retina. J Anat Soc India 2021;70:136-9

How to cite this URL:
Mane P, Sabnis AS. Proliferative capacity of retinal progenitor cells in human fetal retina. J Anat Soc India [serial online] 2021 [cited 2021 Dec 3];70:136-9. Available from: https://www.jasi.org.in/text.asp?2021/70/3/136/326432




  Introduction Top


Retina is a specialized, thin membrane of the eyeball, composed of chain of distinct cells which are involved in receiving light signal from exterior, converting into visual signal, and conveying to brain for perception. Two layers such as neurosensory and pigment epithelium of the retina are derived from the inner and outer part of the neuroectoderm, respectively. Retinal pigment epithelium, supporting cells such as Muller cells and astrocytes, photoreceptors such as rods and cones, supporting neurons such as amacrine and horizontal cells, and neurons such as bipolar and ganglionic cells show a specific arrangement, tremendous coordination for carrying out indispensable function of vision. Damage to any individual neuron within the neural retina could lead to disruption of the retinal function and vision loss.[1] Pathologies of the neural retina represent some of the most common causes of vision impairment and blindness.[2] Retinitis pigmentosa is one of the diseases of the retina where there is degeneration and death of photoreceptor cells, and it affects 1/3000–4000 individuals younger than 60 years of age.[3] Regeneration in retina is possible, which was shown in the 18th and 19th century, and it was confirmed that retinal pigment epithelium could regenerate newt retina.[4] The ciliary marginal zone, retinal pigment epithelium, muller cells, and ciliary epithelial cells have the capacity to reenter the cell cycle, and they express several genes which are expressed in retinal progenitor cells (RPCs).[5] The use of RPCs for neuronal regeneration is an active area of investigation.[6] Progenitor cells in retina are tissue-specific cells which are competent to make cells of desire in retina and this has wide application in retinal transplantation if different retinal disorders. Studies regarding generation of RPCs in rat,[7] xenopus,[8] mice,[9] human,[10] and isolation and efficacy of RPCs,[11] factors involved in differentiation and proliferation,[12] and therapeutic potential of human RPCs[13] have been done. All the dimensions of RPCs studied are of great value. Knowledge of maximum proliferation activity of RPCs in specific gestational age would be considered for retinal transplantation. In the study, proliferative capacity of RPCs in fetal retina is studied in different gestational ages with Ki67 immunohistochemistry marker.


  Material and Methods Top


Type of the study: Exploratory study design

Fifty-six eyeballs (right and left) from 28 fetuses of both sexes of gestational age ranging from 13th to 40th week were obtained from MGM Kalamboli Hospital through spontaneous abortion and medical termination of pregnancy after taking parent's consent and obtaining ethical and scientific approval of institution. Macerated or decomposed fetuses, fetuses with congenital anomalies, and fetuses with a maternal history of any disorders were not included in the study. Fetuses were preserved in 10% formalin. Horizontal and vertical incision was taken on the eyelid and the eyelid was removed. Ten percent neutral buffer formalin was injected around the eyeball. Eyeballs were taken out through the orbit from its anterior aspect with help of special dural forceps. To understand the side of eyeball after enucleation, marking was done with eosin stain on superior and temporal side of eyeball. It was kept in Davidson's fixative solution for 24 h. The eyeballs were cut in horizontal section at the center of cornea and optic nerve projection, and the eyeballs were immersed in 10% neutral formalin for 1 day. Tissue was fixed and treated with immunohistochemistry marker Ki67.

Immunohistochemistry protocol

  1. Section cutting: 3–4 micrometer thick sections of the slides were taken on saline or poly-L-lysine coated slide
  2. It was then transferred to three changes of xylene for 30 min
  3. Later rehydrate with decreasing grades of alcohol absolute, 95%, 70%, and 50%
  4. Finally, the sections were washed under tap water for 30 min
  5. Antigen retrieval


  6. The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagents that break the protein cross-link formed by formalin fixation and thereby uncover hidden antigenic sites.

    This was by adding citrate buffer pH 6 and antigen retrieval reagents. Heat-mediated antigen retrieval was done in the microwave oven at 800 watt for 10 min, following 420 watt for 10 min and 360 watt for 5 min.

  7. Immunostaining


    1. Peroxidase block with 3% hydrogen peroxide in methanol 5 min
    2. Power block: Incubate sections for 10 with primary antibody: anti-CD34/ki-67 (Santa Cruz Biotechnology, Santa Cruz, CA) at 0.025/g/ml
    3. CD34/ki-67 for 30 min at room temperature
    4. Wash in Tris buffer solution pH 7.4 – 10 min
    5. Super enhancer: Incubate with super enhancer for 10 min
    6. Wash in Tris buffer solution pH 7.4 – 10 min
    7. Poly HRP: Incubate with poly HRP for 30 min
    8. Wash in Tris buffer solution pH 7.4 – 10 min
    9. Substrate: Incubate with substrate DAB and check for the color change, brown color appears within 5–10 min
    10. Wash in Tris buffer solution pH 7.4 – 10 min.


  8. Transfer to tap water for 10–20 min
  9. Put into increasing grades of alcohol 50%, 70%, and 95% and absolute alcohol
  10. Transfer to three changes of xylene.


Slides were prepared, dried, mounted in DPX, and covered with coverslip. Seven groups were categorized as per gestational age. Each group was composed of 4 weeks of gestation. They were labelled A to G. the slides were scanned under light microscope at 10 X and 40 X. All the sections were studied under the compound light research microscope and microphotography was done with USB camera.

Data analysis for ki-67: The number of progenitor cells was assessed in sections of the retina. We recorded 20 measurements/fetus. The counts were expressed as average percentage of progenitor cells positive for Ki-67 marker in the retina of each fetus. Then, the mean percentage of progenitor cells was determined for each group.


  Results Top


  1. Observations of right and left retina were similar
  2. Proliferative capacity of RPCs was highest in B group (17–20 weeks) and it was maximum at 19th week of gestation [Table 1].
Table 1: Proliferative capacity of retinal progenitor cells at different groups

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  Discussion Top


Stem cells are the cells which are capable of proliferation, self-renewal, and differentiation into various types of cells. These are called totipotent cells when isolated from fertilized oocyte, pluripotent when isolated from blastocyst, and multipotent when isolated from fully developed adult tissue.[14] Multipotent stem cells are referred as progenitor cells.[15] Ability of progenitor cells to get transform into various cells explores new avenue in medicine where regenerative therapy is essential. Retinal degenerative conditions like retinitis pigmentosa, age related macula degeneration where progressive visual decline results because of continuing loss of photoreceptor cells. Injecting progenitor cells in such cases is a promising attempt which would help to restore vision as progenitor cells have peculiarity to convert into various type of cells of retina. Progenitor cells are able to retain their progenitor status and these cells are not tumorigenic over the period. They may provide renewable, stable, and consistent supply of transplantable cells in the treatment of retinal degeneration.[10] Transplantation of progenitor cells of fetal retina into degenerative retinal diseases would be fruitful in protecting visual function.[13] Human embryonic stem cells can be selectively directed to neural retinal fate and may be useful in the treatment of retinal degeneration.[12] RPCs are obtained from fetal eyes at 16 to 18 weeks of gestation when retinal differentiation has been well defined.[16] Human RPCs from 16th to 18th weeks of gestational age had the longest survival in vitro and yielded the maximum number of cells, proliferating over at least 6 passages. These cells expressed the retinal stem cell markers nestin and Ki-67. They studied the growth of progenitor cells only few weeks from the 12th to 18th weeks of gestational age and showed that cells from donor tissue of 16th –18th weeks of gestational age exhibit the best proliferative dynamics under the specified conditions.[17] After harvesting the cells of the retina, it was found that progenitor cell lines were derived from human fetal retina at 10–12 weeks of gestation and these progenitor cells at 12 weeks are partially committed toward retinal phenotype and competent to develop into cone and rods.[10] In the current study, we observed that maximum proliferation capacity of RPCs is at 19th week [Figure 1] and [Graph 1] and a steady increase in Ki-67 expression between 13th and 20th weeks was detected, which was seen as brown signal with the immunohistochemical marker Ki-67. Ki-67 antibody is a nuclear protein and associated with proliferation of progenitor cells. It is a proliferative marker and is expresses only in cells that are actively dividing.[18] A large subpopulation of human retina progenitor cells showed a characteristic nuclear pattern with Ki-67, indicating that the majority of these cells remained in a proliferative stage. Our findings through immunohistochemistry confirm that progenitor cells sustain their undifferentiated identity in the developing retina. A large population of these undifferentiated cells labeled strongly for Ki-67 at different gestational ages. The molecular and cellular markers, especially cell surface markers, are important tools for evaluating progenitor properties and monitoring the progression of progenitors during development.[19],[20] In the present study, the maximum number of undifferentiated human RPCs were present at the 19th weeks, and afterward, these undifferentiated RPCs get differentiated into mature retinal cell types. Human RPCs can be isolated from fetal human eyes and these cells can be grown in vitro. They also showed that these cells remain in an undifferentiated state through late passage and that they can integrate and differentiate into mature photoreceptors both in vivo and in vitro.[21],[22] The findings of proliferation capacity of RPCs in fetal retina is very promising and it may take us forward in the field of retinal transplantation. Knowledge regarding the highest proliferative phase in fetal retina is favorable to isolate RPCs from ideal gestational age and generate large numbers of RPCs that are genetically and phenotypically similar. This study furnishes the capacity of proliferation of RPCs for marching toward transplantation in the field of regeneration.
Figure 1: Proliferation of retinal progenitor cells in different gestational weeks under ×40. Brown colored signal shows retinal progenitor cell with immunohistochemical marker Ki-67

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  Conclusion Top


Proliferation of progenitor cells in human fetal retina was increasing from 13th to 20th week of gestation with the highest amount of proliferation being at 19th week. To isolate and culture RPCs in human feral retina, 19th week of gestation can be targeted to have fruitful results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Keefe JR. An analysis of urodelian retinal regeneration. I. Studies of the cellular source of retinal regeneration in Notophthalmus viridescens utilizing 3 H-thymidine and colchicine. J Exp Zool 1973;184:185-206.  Back to cited text no. 4
    
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Lamba DA, Karl MO, Ware CB, Reh TA. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A 2006;103:12769-74.  Back to cited text no. 12
    
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Luo J, Baranov P, Patel S, Ouyang H, Quach J, Wu F, et al. Human retinal progenitor cell transplantation preserves vision. J Biol Chem 2014;289:6362-71.  Back to cited text no. 13
    
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Mace KA, Braun KM, editors. Isolation of adult stem cells and their differentiation to Schwann cells. In: Progenitor Cells: Methods and Protocols, Methods in Molecular Biology. Vol. 916., Ch. 5. Humana Press: springer Nature Switzerland Springer-Science and Business Media; 2016. p. 48.  Back to cited text no. 14
    
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Martínez-Cerdeño V, Noctor SC. Neural progenitor cell terminology. Front Neuroanat 2018;12:104.  Back to cited text no. 15
    
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Walcott JC, Provis JM. Müller cells express the neuronal progenitor cell marker nestin in both differentiated and undifferentiated human foetal retina. Clin Exp Ophthalmol 2003;31:246-9.  Back to cited text no. 16
    
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Aftab U, Jiang C, Tucker B, Kim JY, Klassen H, Miljan E, et al. Growth kinetics and transplantation of human retinal progenitor cells. Exp Eye Res 2009;89:301-10.  Back to cited text no. 17
    
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Qu L, Jin X, Chen N, Wang D. Comparison of human retinal progenitor cells cultured in media with or without serum: In vitro and in vivo characteristics and retinal transplantation. Int J Clin Exp Pathol 2018;11:5171-84.  Back to cited text no. 18
    
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Watanabe T, Raff MC. Rod photoreceptor development in vitro: Intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina. Neuron 1990;4:461-7.  Back to cited text no. 19
    
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Altshuler D, Cepko C. A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro. Development 1992;114:947-57.  Back to cited text no. 20
    
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