Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro

Abstract

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C‐terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. Virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10–14 to 10–9 M for MDF1 and 10–15 to 10–5 M for MDF2. Arch. Insect Biochem. Physiol. 40:129–140, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Factors affecting proliferation and differentiation of lepidopteran midgut stem cells

“…Alteration of the calcium titer of the culture medium surrounding midgut stem cells of H. virescens had a profound effect on stem cell fate (Loeb, 2005). Decreasing Ca 21 out by use of the Ca 21 chelating agent ethylene glycol-bis (2-amino ethyl ether)-N, N, N 0 N 0 -tetraacetic acid (EGTA) promoted dose-dependent stem cell multiplication up to 1.5-fold and differentiation to larval forms of midgut epithelium in vitro, similar to that induced by the MDFs (Loeb et al, 1999;Loeb and Jaffe, 2002). In contrast, increasing the concentration of Ca 21 out by adding Ca 21 to the culture medium to a concentration equal to or more than 55 mM, or increasing Ca 21 entry into stem cells by use of the calcium ionophore A23187 (Rosenberger and Triggle, 1978), stimulated cultured larval midgut stem cells to produce scale-like, squamous-like, and vacuolated cells in a dose-dependent fashion, similar to effects of mammalian growth factors PDGF, RA, and EGF.…”

“…A somewhat smaller active peptide of 26 amino acids, HVGKTPIVGQPSIPGGPVRLCPGRIR (Mr 2,689), which we called Midgut Differentiation factor 1 (MDF1), was commercially synthesized (Quality Controlled Biochemicals Inc., Boston, MA). It promoted maximum differentiation of H. virescens larval-type stem cells at concentrations of 10 À14 to 10 À8 M within 7 days (Loeb et al, 1999).…”

“…However, Smagghe et al (2005) showed that ecdysone (E) as well as 20E could serve as a midgut stem cell proliferation agent, although effectiveness was concentration-dependent (Smagghe et al, 2005). 20E was effective only in the absence of juvenile hormone (JH) in stimulating midgut stem cell proliferation in beetle midgut (Tribolium castaneum) in vitro (Parthasarathy Cell-free extracts of fat body (FBX) from pupae of M. sexta and H. virescens were prepared and assayed for effects on highly enriched preparations of stem cells obtained from cultured midgut (Loeb et al, 1999). As titers of FBX were increased, correspondingly more tritiated thymidine was incorporated by the stem cells and was converted to DNA, as evidenced by incorporation of the thymidine analog bromodeoxyuridine (BrdU) (Sadrud-Din et al, 1994;Loeb and Hakim, 1995a), strongly implying that FBX was indeed inducing stem cell multiplication (Fig.…”

“…Over 60% of freshly isolated larval midgut stem cells from H. virescens (approximately 10 mm in diameter) exposed to MDF1 differentiated to larval forms of columnar or goblet cells approximately 30 mm long within 6-8 days (Loeb et al, 1999;Loeb and Jaffe, 2002). In contrast, after 15 days of exposure to platelet-derived growth factor (PDGF), approximately 11% of the cells differentiated to oval cells, approximately 60 mm long, each with a prominent vacuole and no visible microvilli.…”

“…Most remain undefined. Insect growth factors have also been derived from hemolymph and fat body (reviewed in Loeb et al, 1999). Testis sheaths of Lepidoptera release diffusible factors essential to the initiation of meiosis in spermatocytes (Giebultowicz et al, 1987).…”