Cobblestone area measuring (CAM) assay: a new way of assessing the potential of human haemopoietic stem cells.

Abstract

A new in vitro way of scoring the potential (proliferative- differentiative) of human haemopoietic stem cells (HHSC) is presented. We have applied it in the investigation of HHSC behaviour in normal bone marrow specimens under culture conditions. This system describes a modification of some existing culture assays for primitive HHSC function, and proliferation of the cobblestone area forming cell (CAFC) was determined by counting the cobblestone areas (CA) produced every week. The main steps of the assay are: (a) culture of MNC fraction on pre-established fibroblastic confluent layers from the M2.10B4 mouse cell line; (b) weekly counting of CA over a period of 5 weeks; (c) weekly in situ observation of CA morphology; (d) enumeration of secondary progenitors per CA, in the 5th week. The CAM index, expressing the value of the CA kinetic line slope and the number of secondary progenitors per CA were evaluated as factors indicating HHSC proliferative and differentiative potential, respectively. This culture system has the potential to serve as an in vitro assay of human stem cell transplantation. It could be applied to evaluating the potential of HHSC contained in haemopoietic collections of diverse origin, only in combination with measurements of the starting number of CAFC.