Wnt5a Does Not Support Hematopoiesis in Stroma-Free, Serum-Free Cultures

Aneta M Schaap-Oziemlak,Satish Khurana,Catherine M Verfaillie,Sarah Schouteden,Zoran Ivanovic

doi:10.1371/journal.pone.0053669

Aneta M Schaap-Oziemlak, Satish Khurana + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0053669

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Journal: PloS one	Publication Date: Jan 14, 2013
Citations: 17	License type: CC BY 4.0

Affiliation: KU Leuven

Abstract

Previously we reported that Wnt5a is highly expressed in the murine urogenital ridge-derived UG26-1B6 cells but not embryonic liver-derived EL08-1D2 cells. Mouse long-term repopulating hematopoietic stem cells (LTR-HSC) were maintained in non-contact UG26-1B6 cultures but not EL08-1D2 non-contact cultures, unless Wnt5a was also added to the cultures, suggesting a role for Wnt5a in the in vitro maintenance of LTR-HSC. Here, we investigated if the effect of Wnt5a on adult LTR-HSC activity is HSC-autonomous. To test the effect of Wnt5a on maintenance of LTR-HSC, we performed limiting dilution competitive transplantation assays of murine Lin-Sca1+ c-kit+ (LSK) cells cultured for 5 days with TPO and SCF with and without Wnt5a. The effect of Wnt5a on the generation of colony forming units (CFU) and the homing ability of LSK progeny was also tested. No effects were found of Wnt5a on total cell expansion, the number of CFU, or homing ability of day 5 LSK progeny. Furthermore, addition of Wnt5a did not improve, but may have impeded maintenance of LTR-HSC. In conclusion, our data indicate that Wnt5a does not enhance the maintenance and expansion of adult murine LTR-HSCs or committed progenitors cultured in vitro in serum- and stroma-free conditions.

Highlights

Wnt proteins are a large family of secreted glycoproteins that can affect cell fate decisions in many cell compartments
Murine bone marrow (BM)-derived Lin-Sca1+ c-kit+ (LSK) (Lin- Sca-1+ c-kit+) cells were cultured for 5 days in the presence of TPO (100 ng/ml) and Stem Cell Factor (SCF) (50 ng/ ml) alone (TS condition), or with Wnt5a (100 ng/ml) (TSW condition)
The number of colony forming units (CFU)-C in progeny of 50 LSK cells was similar for TSand TSW-treated cells (p = 0.77) (Figure 1B), demonstrating that Wnt5a does not affect hematopoietic progenitor activity in vitro

Summary

Introduction

Wnt proteins are a large family of secreted glycoproteins that can affect cell fate decisions in many cell compartments. Depending on the Wnt protein, the cell type, the type of receptor, the availability of intracellular signalling molecules, signalling from Wnts occurs via the canonical or non-canonical pathways. The non-canonical pathways do not stabilize b-catenin, but cause increases in intracellular Ca2+ levels or activate Jun N-terminal Kinase (JNK) (reviewed in [1]), leading to for instance actin-dependent cytoskeleton reorganisation (reviewed in [2]). As Wnt3a deficiency causes embryonic lethality, HSC in postnatal Wnt3a2/2 mice cannot be studied [10]. Elimination of b-catenin alone or combined with c-catenin in postnatal hematopoietic cells does not lead to impairment in hematopoiesis, suggesting that canonical Wnt signalling does not affect postnatal HSC [11,12]. Due to perinatal lethality of Wnt5a2/2 mice, the in vivo role of Wnt5a in maintenance of adult HSC cannot be studied [13]

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