प्राथमिक मानव तंतुकोशिका संस्कृति से भोले वयस्क स्टेम सेल अलगाव

Summary

हम प्राथमिक मानव fibroblast संस्कृतियों से भोले multipotent त्वचा व्युत्पन्न अग्रदूत (एसकेपी) कोशिकाओं को अलग करने के लिए एक विधि की रिपोर्ट. हम तंतुप्रसू संस्कृतियों से निकाली गई इन SKPs को मानव त्वचा बायोप्सी से सीधे प्राप्त लोगों के लिए समान स्टेम सेल गुण है कि शेयर दिखा. इन कोशिकाओं को इस तरह Oct4 और Nanog रूप multipotent मार्करों के अलावा तंत्रिका शिखा मार्कर, nestin, व्यक्त करते हैं.

Abstract

Over the last decade, several adult stem cell populations have been identified in human skin ^1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory ^{5, 6}. These early studies described a multipotent precursor cell population from adult mammalian dermis ⁵. These cells–termed SKPs, for skin-derived precursors– were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons ⁵. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers ⁶. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.

Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies ⁷. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable ⁷. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures ⁷.

We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Protocol

1. प्राथमिक तंतुकोशिका संस्कृति से एसकेपी अलगाव सेल बैंकों से या सीधे त्वचा बायोप्सी से प्राप्त या तो fibroblast संस्कृतियों fibroblast वृद्धि मध्यम DMEM 15% भ्रूण बछड़ा सीरम, 2 मिमी glutamine, 10 मिलीग्राम / एमएल पेनिसिलिन, ?…

Representative Results

हम हाल ही में 7 की सूचना के रूप में चुनिंदा EGF और FGF2 से मिलकर नियंत्रित विकास शर्त के तहत एसकेपी क्षेत्रों उत्पन्न करने के लिए विस्तार है कि कोशिकाओं की आबादी प्राथमिक त्वचीय fibroblast संस्कृतियों (चित्…

Discussion

विधि का प्रयोग यहाँ बताया, भोले त्वचीय स्टेम कोशिकाओं प्राथमिक त्वचीय fibroblast संस्कृतियों से अलग किया जा सकता है. इस दृष्टिकोण का उपयोग करना, हम हाल ही में एक दुर्लभ आनुवंशिक सिंड्रोम, हचिंसन-Gilford progeria सिंड्?…

Materials

Name of the reagent	Company	Catalogue number	Comments (optional)
DMEM high glucose	Invitrogen	31966-047
DMEM low glucose	Invitrogen	21885-108
fetal bovine serum	Invitrogen	10270-106
L-glutamine	Invitrogen	25030-024	Final conc.: 200 mM
Penicillin/ Streptomycin	Invitrogen	15140-122	Final conc.: 10 mg/ml /10 mg /ml
trypsin solution (0.25%)	Invitrogen	25200-056
F-12 Nutrient Mixture (Ham)	Invitrogen	21765-029
FGF2	BD Biosciences	4114-TC-01M	Final conc.: 40 ng/ml
EGF	BD Biosciences	236-EG-200	Final conc.: 20 ng/ml
PDGFBB	Invitrogen	PHG0043	Final conc.: 5 ng/ml
TGF-b1	Invitrogen	PHG9204	Final conc.: 2.5 ng/ml
25 cm2 flask	Omnilab	FALC353109
PBS w/o CaMg	Invitrogen	14190-169
B27	Invitrogen	17504-044
Methanol	Roth	8388.2
Vectashield mounting medium	Vector Inc.	H-1200
RNeasy Minikit	Qiagen, Valencia, CA	74104
Omniscript Reverse Transcriptase	Qiagen	205113
SsoFast EvaGreen Supermix	BioRad	172-5201
Fungizone	Invitrogen	15290-018	Final conc.:1 mg/ml
			Table 2. Specific reagents and equipment.