Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.
Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells’ (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.
Eski büyüyen bir kaçınılmaz sonucu işlevsel kalır dokuların ve organların azalan yeteneğidir. Yetişkin kök hücreleri, ancak yaş organizmalar gibi, kök hücreler de kendi biyolojik davranışı bir düşüş yaşamaya, doku ve organ homeostasis işlevselliğini korumak için gereklidir. Bu, bağırsak epiteli gibi yüksek devir oranına sahip dokular, özellikle zararlıdır. Yaşlı kök hücrelerin diğerlerinden ayıran genomik hasar, bozulma onarım mekanizmaları, bozulmuş hücre döngüsü düzenleme ve (1-3 gözden), normal kök hücre davranışını etkilemekte olan düzeni bozulmuş sinyal yollarını içerir. Belirli sinyal yolları manipüle veya belirli genleri aşağı vurma, biz düzenleyen ve normal kök hücre davranışlarını muhafaza rolleri içgörü kazanmıştır. Bu deneylerin en adayı molekülü yaklaşımlar olduğundan, biz sırasında kök hücrelerinde meydana endojen moleküler değişiklikler hakkında çok az bilgiye sahipyaşlanma. Bu soruyu yaklaşım bir yolu eski kök hücreler olan ifade profili yaşlanma sırasında önemli ölçüde değişir molekülleri tanımlamak için karşı genç Transcriptome karşılaştırmaktır. Ne yazık ki, biyolojik ve teknik sorunlar bugüne kadar bu yaklaşımı ile ilgili bizim ilerleme engel olmuştur.
Drosophila melanogaster o (4 gözden) fenotipleri yaşlanma kısa bir ömrü (yaklaşık 60-70 gün) ve sergiler beri yaşlanma incelemek için son derece uygun bir model organizmadır. Ayrıca, Drosophila yaşlanma işlem ısı derecesi ile hızlandırılabilir. 29 ° C 'de sinekler korurken, bağırsak dokusunda yaşlı fenotip önce 15 gün 5 fark edilebilir. Ayrıca, Drosophila genetik manipülasyonlar bir bolluk için elverişlidir. Özellikle, Drosophila orta bağırsak, farklı sinyal yolları ve çevre sorunlar etkisini incelemek için mükemmel bir model sistem olarak ortaya çıkmıştır(10/06 gözden) yaşlanma sırasında bağırsak kök hücreleri (ISCs) biyolojisi. Drosophila bağırsak epitel yüksek ciro oranına sahip ve erkeklerde 11 ayda bir kez kadınlarda her iki haftada bir yenilenir ve. Drosophila midgut ikamet eden ISCs bölmek ve kendi kendine yenilenen ISC ve enteroblast (EB) 12,13 olarak adlandırılan bir post-mitotik progenitör hücre üretme kapasitesine sahip. EB emici bir enterosit veya salgı enteroendokrin hücreye ya belirlemektedir. Bu güne kadar, açıkça bir ISC etiketler tek işaretleyici kombinasyonu transkripsiyon faktörü salyangoz (esg) ve Çentik ligandı Delta (Dİ) 14 ifadesidir. Bununla birlikte, bu, sadece genç ve sağlıklı bir bağırsak için de geçerlidir. Yaşlanma sırasında, orta bağırsak epitel ISC çoğalması 15-17 bir artış ile karakterize edilmektedir. Ayrıca, anormal Notch sinyal ISC kızı hücrelerinin kaderi kararını bozanve EBS 15 misdifferentiation neden olur. Bu sayede bir yaşlı midgut niyetli kök hücreleri tanımlamak için yetersiz Dİ ifade render, Notch sinyalizasyon ve ko-ekspres ESG ve Dİ için aktif hücrelerin birikmesi ile sonuçlanır. Gerçek ISCs tanımlamada zorluk şimdiye kadar yaşlanma ISCs endojen değişiklikleri incelemek için yeteneği engellemiştir.
Biz ISCs ve EBS GFP seviyesi doğal olarak farklı ve yaşlanma boyunca farklı kaldığı ESG -Gal4, UAS-GFP transgenik sinek hattı yararlanarak bu sorunu ele oylandı. Benzer bir yaklaşım, larva nöroblast- ve nöronlar 18,19 izolasyonu için tarif edilmiştir. Genç ve yaşlı ESG -Gal4, UAS-GFP sinek Midguts disseke ve tek hücre içine ayrışmış edildi. Hücreler daha sonra, floresan aktive hücre sınıflandırma (FACS) kullanılarak GFP-pozitif hücreler için saklanmıştır. İlginçtir, sıralanmış GFP pozitif cGFP floresan yoğunluğuna dayalı iki ayrı zirveleri dağıtıldı arşın (GFP yüksek ve GFP düşük). Ayrıca, aynı zamanda, hücre boyutu ile ilişkili iki adet tepe noktasına GFP pozitif hücrelerin dağılımı: düşük GFP yoğunluğu sergiledi hücrelerin küçük ve yüksek GFP yoğunluğuna sahip hücreleri ise daha az zerre daha büyük ve daha granüler idi. Bu gözlem küçük ISCs GFP yoğunluğuna dayalı FACS kullanarak ve uygun ileri dağılım (FSC) ve yan dağılım (SSC) ayarları seçerek büyük EBS ayırt edilebileceğini önerdi. Ilginç bir iki tepe belirgin yaşlanma sırasında ayrılmış kaldı. Ayrıca, iki adet tepe oranı, yaşlanma midguts bahsedilen ayırt edici yansıtan bir şekilde değiştirildi: büyük sayısı, zaman içinde EBS artar misdifferentiated yani bu. Bu bulgular biz uygun FACS parametre ayarlarını kullanarak GFP pozitif hücreler için sıralama biz her zaman noktası d ISCs ve EBS için zenginleştirebilirsiniz sonucuna varıldırada yaşlanma.
Özetle, biz herhangi bir yaş Drosophila midguts gelen, iki farklı hücre popülasyonlarının, ISCs ve EBS için zenginleştirmek ve gelecek nesil dizileme gibi daha fazla analiz için bu hücrelerin, izole etmek araştırmacıların sağlayan bir FACS stratejisi tanıtmak. Bu güçlü bir yöntem kök veya projenitör hücreler, zenginleştirilmiş bir popülasyonda yaşlanma doğasında olan endojen molekül mekanizmalarının incelenmesi için izin verir. Bu çalışmalardan elde edilen veriler, kuşkusuz türler arasında yaşlanan önemli olan korunmuş moleküllerin belirlenmesini kolaylaştırır.
The presented protocol describes a method to isolate ISCs from young and old adult Drosophila midguts, which can subsequently be used for further molecular analyses such as next generation sequencing. FAC sorting of the GFP-positive cell population from the esg-Gal4, UAS-GFP fly line has already been achieved by several groups21,22. However, until now the presence of the two distinct peaks of GFP-positive cells has been either overlooked or undervalued. We show that this separation not only represents two different populations of cell types (ISCs and EBs), but also that the two peaks of GFP-positive cells stay distinctly separate during aging. This observation is highly relevant and valuable to researchers interested in studying stem or progenitor cells during aging. Isolation of ISCs from old midguts has been hampered by the fact that there is no molecular marker combination to identify ISCs in an aged midgut. The only marker that unambiguously identifies ISCs in an old midgut is phospho-histone3 (PH3), since ISCs are the only dividing cells in the midgut12,13. However, PH3 is an unsuitable marker to isolate ISCs since the number of dividing stem cells at any given time point is too low in order to obtain a decent amount of ISCs for subsequent analyses. The fly line esg-Gal4, UAS-GFP is the most common fly line used by researchers who study ISC function, maintenance and differentiation. Our current knowledge on the molecular regulation of ISCs homeostasis is based on studies in which specific molecules and signaling pathways have been manipulated (reviewed in 7). Hence, we still lack knowledge about the endogenous molecular changes that occur during aging. The herein described method closes this gap and is based on the fact that ISCs can be isolated based on their size, granularity and GFP intensity from adult midguts at any time point during aging.
While establishing and optimizing this method we have found the following steps to be critical for a reliable outcome. Approximately 200 guts need to be dissected in order to obtain a good amount of ISCs for FAC sorting. The speed of dissection is crucial and depends on the individual’s practice. A trained individual can dissect 40 guts within 20–30 min. Since GFP is also expressed in the Malpighian tubules in the esg-Gal4, UAS-GFP fly line, it is essential to completely remove the Malpighian tubules from the midgut. The dissected midguts must be kept in a cool environment to avoid tissue degradation and reduce RNAse activity in the tissue. Therefore, the dissected midguts must be transferred from the dissecting dish into microcentrifuge tubes containing cold 1x PBS/1% BSA solution after a maximum of 20-30 min and kept on ice. However, the dissected midguts should not be left on ice for more than 2 hr before starting the tissue dissociation with trypsin. The most efficient approach is to dissect as many batches of flies as possible within these 2 hr, then start the trypsin digest for these batches. If more midguts are needed, they can be dissected during the incubation times of the trypsin digest.
Although trypsin is a very potent enzyme and could have detrimental effects on cells, we still obtained enough living, healthy cells for subsequent FAC sorting. The key step of our procedure that allows for the isolation of intact cells is that the dissociated cells are removed from the trypsin solution every 30 min. If the dissected midguts are incubated for 2 ½ hr in a trypsin containing solution at room temperature, we observed a 20–30% increase in dead, Sytox-positive cells. It should be pointed out that the trypsin solution we use contains EDTA. The presence of EDTA probably facilitates tissue disintegration by chelating calcium and magnesium ions needed for the proper function of extracellular matrix molecules.
The most crucial steps to successfully sort ISCs from young and old guts are the appropriate initial settings of the FACS and gating parameters using the described controls and following a specific sorting hierarchy. We performed the FAC sorting on an ARIA II Flow Cytometer (FACSDiva software). It is important that the instrument is turned on at least one hour prior to sorting to warm up the lasers. Of note, when FAC sorting of ISCs is performed for the first time, the parameters for sorting and for compensation need to be set using the aforementioned controls: (1) dissociated cells from wild type (e.g. w1118) Drosophila midguts without addition of Sytox — setting this parameter (Step 4.4.1) prevents sorting cells based on their autofluorescence; (2) dissociated cells from wild type (e.g. w1118) Drosophila midguts with Sytox added — setting this parameter (Step 4.4.2) allows separating dead from living cells (dead cells have a high autofluorescence and can contaminate the sorted GFP-positive cells); (3) dissociated cells from midguts of the esg-Gal4, UAS-GFP fly line without Sytox — setting this parameter (Step 4.4.3) ensures that all GFP-positive cells are plotted within the scatter plot. If these parameters are not set properly, the sorted cell population will most likely contain dead cells and debris (Figure 5B, C), many GFP-positive cells will be missed (Figure 5D) and the two distinct peaks for GFP-positive cells as seen in the histogram plot (Figure 2E and Figure 3E) will not be detected. Once the FACS and gating parameters have been set, the instrument is calibrated and ready to sort cells from the esg-GAL4, UAS-GFP fly line. Since these parameters can be saved, all future sorting sessions for ISCs and EBs from the esg-GAL4, UAS-GFP fly line can start from Step 4.5. Although choosing the “Purity” mode and performing a two-way purity sort decreases the number of cells sorted, it allows for a higher sorting stringency (Step 4.6 and Figure 4B, C). Of note, the advantage of using Sytox instead of propidium iodide to label dead cells is that there is only a low spillover into the FITC (GFP) channel, which makes it easy to compensate for the spillover.
Our method of enriching for ISCs and EBs, respectively, is based on sorting the cells according to different GFP expression levels. It may be that during aging the misdifferentiated EBs also express GFP at a lower level and could be mistaken as ISCs. However, since the sorted cells also differ in size and granularity, we believe that our sorting strategy is the most suitable to this date to enrich for ISCs and EBs. Also, it is known that cells in an aging midgut retaining ISC identity have a small nucleus15. To obtain more clarity on the identity of the sorted cells, one could sort cells from a transgenic fly line that carries esg-GAL4, UAS-GFP and a Notch signaling reporter transgene. Since Notch has been shown to be active only in the EBs12,13, ISCs isolated from a young midgut would be negative for the Notch reporter, whereas the EBs would be positive for the Notch reporter. However, during aging Notch signaling becomes aberrant in the midgut tissue. Therefore, it needs to be tested whether this experimental set up is indeed more reliable to distinguish between ISCs and EBs isolated from an old midgut.
We have already employed this approach for comparative transcriptome analysis of ISCs from young and old midguts and identified a number of factors that are differentially regulated during aging (unpub. observ.). Additionally, this method allows for analyzing differences in gene expression between ISCs and the EBs. Such investigations will provide insight into the initial molecular changes that occur as a cell enters the differentiation process. Also, the isolation of EBs during aging and subsequent analyses will shed light on the molecular changes of age-induced misdifferentiation. Furthermore, this method can be combined with other genetic tools used in Drosophila to study gene function. In summary, this method offers an unbiased approach to investigate molecular characteristics and changes of ISCs and EBs during aging. This is a valuable tool that will facilitate exploration of aging mechanisms in adult stem and progenitor cells.
The authors have nothing to disclose.
We are grateful to Gabriele Allies for excellent technical assistance. We thank the University Ulm Medical Faculty for the use of the FACS Core Facility and the Institut für Molekulare und Zelluläre Anatomie for using the confocal microscope. We thank S. Hayashi for the esg-Gal4, UAS-GFP fly line. This project is funded by the Federal Ministry of Education and Research (BMBF, Forschungskern SyStaR). A.T. is supported by SFB 1074 (Project A2). A.T. and G.A. are supported by the Deutsche Forschungsgemeinschaft (DFG, FE578/3-1). H.M.T. is a member of the International Graduate School in Molecular Medicine Ulm (GSC 270).
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Forceps: Dumont, Inox Biologie #5 | Fine Science Tools | 11252-20 | |
SefarNitex 03-150um/38 (35 µm nylon mesh) | Sefar | 3A03-0150-102-00 | |
Falcon 5ml Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap | Corning | 352235 | |
Polymax 1040 | Heidolph | 543-42210-00 | |
Albumin from bovine serum (BSA) | Sigma | A4503-50G | |
0.5% Trypsin-EDTA | Invitrogen | 15400-054 | Trypsin obtained from a different company most likely has a different activity and the duration of the trypsin digest has to be adjusted accordingly. |
SYTOX Blue Dead Cell Stain for flow cytometry | Life Technologies | S34857 | |
RNAlater Stabilization Solution | Life Technologies | AM7023 | other solutions, e.g. Trizol can be used for subsequent RNA isolation |
FACSAria II cell sorter | Becton Dickinson | Turn on one hour prior to sorting |