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

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

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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