Analysis of Aged Dysfunctional Intestinal Stem Cells.

Aging is a multifactorial process. Organ maintenance and tissue regeneration are impaired upon aging mainly due to loss of stem cell function in organs that depend on stem cell in the adult. Intestine is such an organ, and upon aging intestinal regeneration is impaired due to decline of intestinal stem cell function. To determine the aging status of intestine and intestinal stem cells, histological analyses; analyses of the level of proliferation markers in tissue by immunofluorescence and/or quantitative RT-PCR; and gene expression analysis for stemness related genes in isolated crypts, intestinal stem cells (ISC), and Paneth cells can be used. To analyze the level of regeneration in intestine and thus determine a decline in ISC function, techniques like in vitro organoid cultures and lineage tracing with BrdU, lineage tracing using transgenic mice and histological analyses of tissue regeneration after 3 and 5 days after two rounds of 10 Gy of radiation (a 10 + 10 Gy IR experiment) can be applied. In this chapter we will focus on protocols for lineage tracing, the 10 + 10 gy IR experiment and for organoid cultures from young and aged mouse intestine.Lineage tracing experiments in intestine can be done in many ways. In this chapter we describe a protocol for lineage tracing upon BrdU incorporation and lineage tracing using the Lgr5eGFPCreERT2 Rosa26YFP transgenic mouse. For BrdU based-lineage tracing BrdU is administrated via intraperitoneal injections into mice. Animals will be analyzed 3 days (72 h) after BrdU administration. For experiments involving Lgr5eGFPCreERT2 Rosa26YFP mice, mice will be analyzed after tamoxifen injection that activates Cre in Lgr5 positive (ISC) cells, which will result in permanent YFP expression. This allows for tracing of YFP positive cells in the intestine. The time point for the analysis of the intestinal tissue will depend in this case on the underlying scientific question that will be addressed. For 10 + 10 Gy experiments, animals will be irradiated with a radiation dose of 10 Gy on 2 consecutive days. The intestinal tissue will be analyzed 3 and 5 days after the second dose of radiation. Quantitative analyses of crypt depth and determination of the rate of crypt fission upon histochemistry will provide an estimation on the in vivo regenerative potential of ISCs. For serial organoid culture experiments, crypts will be harvested from mouse intestine, initially plated at concentrations ranging from 500 to 1000 crypts per well in Matrigel and grown in conditional medium or ISC medium. ISC Medium is changed every 2 days. After 1 week in culture, the organoids will be disrupted via a syringe and replated in fresh Matrigel. As the ability to form multilobed organoids is considered to be a direct stem cell function, the frequency of organoid formation in serial replating experiments can serve as a quantitative measurement of ISC function. For example, we demonstrated a reduced frequency of organoid formation as well as a reduction in number of lobes formed per organoid after 4-5 replatings of intestinal organoids from aged compared to young mice. These three techniques are thus, in combination, able to quantify the regeneration potential of intestinal stem cells and thus determine the extent to which intestinal stem cell regenerative function is reduced upon aging.