Human bone marrow mesenchymal progenitors: perspectives on an optimized in vitro manipulation

Abstract

When it comes to regenerative medicine, mesenchymal stem cells (MSCs) are considered one of the most promising cell types for use in many cell therapies and bioengineering protocols. The International Society of Cellular Therapy recommended minimal criteria for defining multipotential MSC is based on adhesion and multipotency in vitro, and the presence or absence of select surface markers. Though these criteria help minimize discrepancies and allow some comparisons of data generated in different laboratories, the conditions in which cells are isolated and expanded are often not considered. Herein, we propose and recommend a few procedures to be followed to facilitate the establishment of quality control standards when working with mesenchymal progenitors isolation and expansion. Following these procedures, the classic Colony-Forming Unit-Fibroblast (CFU-f) assay is revisited and three major topics are considered to define conditions and to assist on protocol optimization and data interpretation. We envision that the creation of a guideline will help in the identification and isolation of long-term stem cells and short-term progenitors to better explore their regenerative potential for multiple therapeutic purposes.

Keywords: bone marrow; colony; in vitro expansion; mesenchymal stem cell; mutipotent progenitor.

Figure 1

Schematic representation of the suggested correlation between the mesenchymal stem cell differentiation cascade and colony size in vitro. It is frequently inferred that most primitive progenitors give rise to larger colonies compared to those originated from intermediate and committed progenitors.

Figure 2

Two hypotheses discussing the potential differences in the Colony Forming Efficiency results associated with the usage of different fetal bovine serum lots. The “Simultaneous Growth Hypothesis” suggests colonies are similarly affected and respond simultaneously, proliferating more or less, but in the same proportion. The “Selective Growth Hypothesis,” suggests that in response to different FBS lots, a few progenitors will proliferate more than others, as a result of growth factors concentration and combination.

Figure 3

It is generally accepted that most primitive progenitors, upon exiting the lag growth phase, will reach higher proliferation rates than committed progenitors. However, recent data indicate that a few progenitors might require a longer time to enter replicative cycles in vitro. We and others hypothesize that, if cultured longer, these progenitors might originate larger colonies.

Figure 4

In order to establish a “surveillance system” when working with mesenchymal progenitors isolation and expansion, it is suggested that each laboratory keep a standard collection of bone marrow samples to be used as references for testing serum lots. Conceptually, each lab could select representative bone marrow samples, split each of them into equal aliquots (10, 20, 30, or as many as possible), freeze all at the same time, and cryopreserve. These aliquots will then be thawed and used to test serum “quality” every time changes of lots are necessary. From time to time, bone marrow collection must be renewed.

Figure 5

FBS influence on progenitor cell adhesion in vitro must be tested. In group (A), bone marrow cells are incubated in culture medium supplemented with control serum. After 72 h, non-adherent cells are discarded and adherent cells are cultured for 10 additional days also in culture medium supplemented with control FBS. In group (B), bone marrow cells are incubated in culture medium supplemented with testing serum. After 72 h, non-adherent cells are discarded and adherent cells are culture for 10 additional days in culture medium supplemented with control FBS. Colony numbers and size must be evaluated.

Figure 6

Secondary colonies assay. Bone marrow cells are incubated in culture medium supplemented with control or testing FBS. Seventy-two hours later, non-adherent cells are discarded and adherent cells are cultured in culture medium supplemented with control or testing serum for 10 additional days. Later, cells are tripsinized and single-cell suspension prepared. One thousand cells are subsequently replated and incubated for 10 additional days in culture medium supplemented with testing or control serum. Numbers and size of secondary colonies must be evaluated.