Behavior in culture A
cobblestone area-forming cell (CAFC) assay is a cell culture-based empirical assay. When plated onto a confluent culture of stromal
feeder layer, a fraction of hematopoietic stem cells creep between the gaps (even though the stromal cells are touching each other) and eventually settle between the stromal cells and the substratum (here the dish surface) or trapped in the cellular processes between the stromal cells.
Emperipolesis is the
in vivo phenomenon in which one cell is completely engulfed into another (e.g.
thymocytes into
thymic nurse cells); on the other hand, when
in vitro, lymphoid lineage cells creep beneath
nurse-like cells, the process is called
pseudoemperipolesis. This similar phenomenon is more commonly known in the HSC field by the cell culture terminology
cobble stone area-forming cells (CAFC), which means areas or clusters of cells look dull
cobblestone-like under phase contrast microscopy, compared to the other hematopoietic stem cells, which are refractile. This happens because the cells that are floating loosely on top of the stromal cells are spherical and thus refractile. However, the cells that creep beneath the stromal cells are flattened and, thus, not refractile. The mechanism of pseudoemperipolesis is only recently coming to light. It may be mediated by interaction through
CXCR4 (CD184) the receptor for CXC Chemokines (e.g.,
SDF1) and
α4β1 integrins.
Repopulation kinetics Hematopoietic stem cells (HSC) cannot be easily observed directly, and, therefore, their behaviors need to be inferred indirectly. Clonal studies are likely the closest technique for single cell in vivo studies of HSCs. Here, sophisticated experimental and statistical methods are used to ascertain that, with a high probability, a single HSC is contained in a transplant administered to a lethally irradiated host. The clonal expansion of this stem cell can then be observed over time by monitoring the percent donor-type cells in blood as the host is reconstituted. The resulting time series is defined as the repopulation kinetic of the HSC. The reconstitution kinetics are very heterogeneous. However, using
symbolic dynamics, one can show that they fall into a limited number of classes. To prove this, several hundred experimental repopulation kinetics from clonal Thy-1lo SCA-1+ lin−(B220, CD4, CD8, Gr-1, Mac-1 and Ter-119) c-kit+ HSC were translated into symbolic sequences by assigning the symbols "+", "-", "~" whenever two successive measurements of the percent donor-type cells have a positive, negative, or unchanged slope, respectively. By using the
Hamming distance, the repopulation patterns were subjected to cluster analysis yielding 16 distinct groups of kinetics. To finish the empirical proof, the
Laplace add-one approach was used to determine that the probability of finding kinetics not contained in these 16 groups is very small. By corollary, this result shows that the hematopoietic stem cell compartment is also heterogeneous by dynamical criteria. It was originally believed that all hematopoietic stem cells were alike in their self-renewal and differentiation abilities. This view was first challenged by the 2002 discovery by the
Muller-Sieburg group in San Diego, who illustrated that different stem cells can show distinct repopulation patterns that are epigenetically predetermined intrinsic properties of clonal
Thy-1lo Sca-1+ lin−
c-kit+ HSC. The results of these clonal studies led to the notion of
lineage bias. Using the ratio \rho = L/M of lymphoid (L) to myeloid (M) cells in blood as a quantitative marker, the stem cell compartment can be split into three categories of HSC.
Balanced (Bala) hematopoietic stem cells repopulate peripheral white blood cells in the same ratio of myeloid to lymphoid cells as seen in unmanipulated mice (on average about 15% myeloid and 85% lymphoid cells, or 3 ≤ ρ ≤ 10).
Myeloid-biased (My-bi) hematopoietic stem cells give rise to very few lymphocytes resulting in ratios 0 10. All three types are normal types of HSC, and they do not represent stages of differentiation. Rather, these are three classes of HSC, each with an epigenetically fixed differentiation program. These studies also showed that lineage bias is not stochastically regulated or dependent on differences in environmental influence. My-bi HSC self-renew longer than balanced or Ly-bi HSC. The myeloid bias results from reduced responsiveness to the lymphopoetin
interleukin 7 (IL-7). For example, the Eaves group confirmed in 2007 that repopulation kinetics, long-term self-renewal capacity, and My-bi and Ly-bi are stably inherited intrinsic HSC properties. In 2010, the Goodell group provided additional insights about the molecular basis of lineage bias in
side population (SP) SCA-1+ lin− c-kit+ HSC. As previously shown for IL-7 signaling, it was found that a member of the
transforming growth factor family (TGF-beta) induces and inhibits the proliferation of My-bi and Ly-bi HSC, respectively. ==Etymology==