Abstract
Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34(+) progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.
Highlights
An important prerequisite for hematopoietic stem and progenitor cells (HSPCs) migration is the acquisition of a polarized morphology through the reorganization of the cytoskeleton elements leading to the formation of a lamellipodium at the front side and a uropod at the rear [2]
The Rho Kinase Pathway Is Involved in the Formation and/or Stabilization of Uropod and Microvillar-like Structures of Hematopoietic Progenitors—Primary human HSPCs have been described to have various morphologies upon cultivation on mesenchymal stromal cells (MSCs) [25]
Ϸ40% of HSPCs displayed a migrating morphology within the first 4 days, whereas this percentage decreased to 10 –15% after 8 days. The integrity of these plasma membrane protrusions appeared dependent on the Rho kinase pathway, because the incubation of HSPCs with Rho kinase inhibitor Y-27632 resulted in a dramatic alteration of the referred morphologies
Summary
An important prerequisite for HSPC migration is the acquisition of a polarized morphology through the reorganization of the cytoskeleton elements leading to the formation of a lamellipodium at the front side and a uropod at the rear [2]. The Rho Kinase Pathway Is Involved in the Formation and/or Stabilization of Uropod and Microvillar-like Structures of Hematopoietic Progenitors—Primary human HSPCs have been described to have various morphologies upon cultivation on MSCs [25].
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