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. 2005 Sep 1;106(5):1574-80.
doi: 10.1182/blood-2005-03-1017. Epub 2005 May 3.

A novel stem-cell population in adult liver with potent hematopoietic-reconstitution activity

Darrell N Kotton  1 , Attila J Fabian, Richard C Mulligan
Affiliations

A novel stem-cell population in adult liver with potent hematopoietic-reconstitution activity

Darrell N Kotton et al. Blood. .

Abstract

A number of recent reports have documented that cells possessing hematopoietic-reconstitution ability can be identified and isolated from a variety of solid organs in the adult animal. In all studies to date, however, purified organ-derived stem cells demonstrate a diminished repopulating capacity relative to that of purified bone marrow-derived hematopoietic stem cells (BM HSCs). It has therefore been unclear whether organ-derived HSCs possess functional properties distinct from those of BM HSCs, or simply have not been purified to a comparable extent. Here we report the identification of a rare subset of cells in adult murine liver that possess potent blood-repopulating potential, approaching that of BM HSCs. The cells, isolated on the basis of dye-efflux activity and CD45 expression (termed CD45(+) liver side population [SP] tip cells), exhibit a surface phenotype similar to that of freshly isolated BM HSCs derived from normal adult animals, but are phenotypically distinct in that they do not express the stem-cell marker c-kit. Single-cell transplantation studies indicate that CD45(+) liver SP tip cells can be generated from BM HSCs, suggesting a relationship between stem-cell populations in the liver and bone marrow compartments. Overall, these studies have important implications for understanding extramedullary hematopoiesis, and may be relevant to current strategies aimed at inducing tolerance to transplanted organs.

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Figures

Figure 1.
Figure 1.
Adult liver and bone marrow SP cells and identification of the liver CD45+ SP tip. FACS analysis of Hoechst-stained single-cell suspensions identifies a side population (SP) of cells in murine adult liver (A), Ficolled adult liver (B), and Ficolled bone marrow (C). Closer inspection of the dot plot of liver SP shown in panel B reveals a cluster of SP cells with highly efficient dye-efflux properties (termed SP tip cells) that is distinct from cells found higher in the SP gate (SP shoulder cells; gates from B are redrawn in D to show cell frequencies of tip and shoulder subpopulations). Hoechst dye efflux in SP cells is inhibited by verapamil (E). Frequencies of cells expressing the panhematopoietic surface marker, CD45, in liver SP tip, shoulder, and non-SP cells are shown in panels F, G, and H, respectively. Rare CD45+ cells, representing just 3% of SP tip cells, are identified in panel F. Numbers in all graphs represent percentage of events contained in the illustrated gate.
Figure 2.
Figure 2.
Kinetics of competitive blood repopulation after transplantation of SP cells purified from adult bone marrow, liver, or peripheral blood. Purified cell populations from CD45.1 mice were transplanted in competition with 2 × 105 unfractionated bone marrow cells into lethally irradiated CD45.2 mice. Average peripheral blood chimerism arising from the donor test cells was followed monthly. Two hundred liver SP (LiSP) tip CD45+ cells demonstrated potent hematopoietic-repopulation activity that approached bone marrow (BM) SP (P = .08). In contrast, nucleated peripheral blood cells, blood SP cells, and LiSP shoulder CD45+ cells possess limited, if any, competitive repopulation potential compared with bone marrow SP (P < .05) (n = 4 per group per experiment repeated twice; average chimerism at 3 months is enumerated above each bar; bars represent mean chimerism and error bars show SEM).
Figure 3.
Figure 3.
Peripheral blood chimerism after competitive transplantation of limiting dilutions of CD45+ liver SP tip cells. (A) Average 3-month blood chimerism arising from competitive transplantation of 200, 100, 50, or 10 purified CD45+ liver SP tip cells (LiSP) (n = 4 per group; error bars represent SEM). (B) Peripheral blood FACS analysis from a recipient in each group. CD45.1+/CD45.2– events illustrate long-term blood reconstitution from varying doses of purified liver-derived cells. CD45.2+/CD45.1– events arise from 2 × 105 transplanted unfractionated competitor bone marrow cells or residual recipient cells that survived lethal irradiation. Numbers in dot plots represent percentage of cells within each gate.
Figure 4.
Figure 4.
Multilineage blood differentiation 7 months after transplantation. (A) Peripheral blood chimerism, measured by FACS, is shown arising from 200 transplanted liver CD45+ SP tip cells (from a CD45.1 donor) over a 7-month period (n = 4 mice per group per experiment, repeated twice; error bars reflect standard deviation of the mean). (B) Representative FACS analysis of peripheral blood at 7 months shows robust engraftment of differentiated lymphoid (CD3+ or B220+), myeloid (CD11b+ or GR-1+), and erythroid (Ter-119+) hematopoietic lineages derived from the transplanted donor (CD45.1+) cells. Numbers in dot plots represent percentage of cells within each quadrant.
Figure 5.
Figure 5.
CD45+ SP tip cells from liver (Li) are phenotypically distinct from bone marrow (BM) SP cells. (A) Effects of enzyme digestion (collagenase and dispase) on c-kit immunostaining in BM SP cells. Despite diminished intensity of c-kit surface immunostaining after enzyme digestion, 61% of BM SP cells still exhibit c-kit immunofluorescence above background. (B) After identical enzyme digestion, c-kit immunostaining is readily detected above background in CD45-Li non-SP cells and minimally detected in few CD45-LiSP cells. In contrast, no CD45+ SP tip cells from liver exhibit c-kit surface staining. IgG indicates immunoglobulin G. (C) Multiplex RT-PCR assay for c-kit and GAPDH gene expression shows no detectable c-kit mRNA in multiple sorted samples of CD45+ LiSP tip cells. In contrast, c-kit expression is easily detected in samples prepared from identical numbers of sorted BM SP and BM main population (MP). Expression of the housekeeping gene GAPDH is present in all samples. Numbers in dot plots represent percentage of cells contained in each quadrant.
Figure 6.
Figure 6.
Liver CD45+ SP tip cells can be generated from bone marrow (BM) SP or single stem cells after transplantation. FACS analysis of liver (Li) SP tip cells in lethally irradiated recipients that were transplanted with (A) 2 × 106 unfractionated GFP+ marrow, (B) 200 purified bone marrow (BM) SP, or (C) single BM SP cells. CD45+ Li SP tip cells derived, in part, from donor (GFP+) marrow cells in each recipient. (D) CD45+ Li non-SP cells also derived from the transplanted single BM SP cell. Numbers in dot plots represent percentage of cells contained in each gate.
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