IMMUNOHISTOCHEMICAL ANALYSIS OF BASEMENT MEMBRANE FORMATION IN EMBRYOID BODIES DERIVED FROM EMBRYONIC STEM CELLS

Year 2001, Volume: 54 Issue: 3, 167 - 175, 30.09.2001

Hafize Seda Vatansever

Abstract

The central role of basement membranes in organ and cell biology has made them an important subject for researchers who are investigating their structure, organi- satión and regulatory íuncrions. Our study employed indirect immunohístochemistry to investi ate the forma- tion of basement membranes in embryonic bodies (EB) derived from embryonic stem (ES) cells. Basement mem- branes initially appeared in embryonic bodies at the end of the first day. Depositions of basement membrane components - laminin, collagen I\/, nidogen and per- lecan - appeared in cultures as linear staining. Thick basement membrane depositions were also observed in embryonic bodies at the end of the first week using anti- laminin, anti-collagen IV, anti-nidogen and anti-perlecan antibodies. Using TEM, the u/trasfrucrure and thickness of the basement membrane was observed to be similar to Reichert's membrane, a membrane consisting of parietal endoderm cells derived from primitive endoderm cells, secrete basement membrane components and the tro- phectoderm basement membrane. However, ES cells, which are derived from inner cell mass cells, cannof be differentiated from trophoectoderm cells; therefore, basement membranes in EBs may form mechanisms other than Reichert's membrane. It is likely that different basement membrane types are present in EBs čiot cannot be distinguished by immunohistochemistry.

Keywords

: Basement Membrane , Embryonic Stem Cells , Immunohistochemistry

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EMBRiYONiK KÖK HÜCRE1ERiNDEN FARKLlLA- NAN EMBRiYOBLASTLARDA CELiŞEN BASAL MEMB- fłAMtN iMMUMOHiSTOKiMYASAL OLARAK iNCE- LENMESi

Abstract

Organ ve dokulardaki hücrenin temel biyolojik fonksi- yonlarinda önemli rol oynayan basal membranin, yapisi, organizasyonu ve reg”ülasyonu gün 'ûm'üzdeki araştirma konularindandir. Bu nedenle, embriyonik kök hücrele- rinden gelişen embriyoblastlardan o/uşan basal membra- nin yapisi elektron mikroskobik ve indirekt immunohis- tokimya teknikleri ile incelenmiştir. Basal membranin formasyonu ilk olarak 1 @ünl”ük kültür yapilmiş embri- yoblastlarda gözlendi. Basal membran proteinleri olan laminin, kollagen IV, nidogen ve perlekanin immunore- aktivitelerinin |oozitífliği bi‘rg'ûnlük kûltürde bir çizgi şek- linde sa¡otandi. Embriyoblastlarin 1 haha sonraki k'ültür dõnemlerinin analizlerin de ise, basal membranin kalin bir tabaka olarak laminin, żo//agen IV, nidogen ve ¡oerle- kan antikorlari ile immunohistokimya tekniği ile boyan- dim göz/en¢/i. Yapisal ve kalinlik açisindan embriyob- lastlarda farklilanan basal membranin TEM yöntemi ile Reichert'in membranina benzediği gõzlendi. Reichert'in membrani primitíf endoderm h”ücrelerinden farklilanan ve basal membran [:iroteinIerini salgilayan parietal hüc- reler (Smith and Strickland, f98f; Hogan et a/., 198 I; Dziadek et al., 1985; Paulsson et al., 1 985) ve trofoekto- derm h'ücrelerinin basal membrani (Hogan et al., 1984,• Salamat et at., 1995) tarafindan oluşturulur. Bununla be- raber, embriyonun iç h”ücre kitlesinden faklilanan embri- yonik kök h”ücreleri trofoektoderm h”ücrelerine faklilana- maz. Bu nedenle, embriyoblastlarda gelişen basal membranin Reichert’ in membraninin ge/işiminden fark- Ii bir mekanizma ile geliştigi d‘dş”ün”ülmektedir. Tabiki, immunohistokimyasal olarak embriyoblastlardan ge/işen basal membranin tipi ayirt edilemez.

Keywords

Basal Membran , Embríyoblast , lm- munohístokimya.

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