Page 127 - Hematology_ Basic Principles and Practice ( PDFDrive )

98 Part II Cellular Basis of Hematology

cell sorting (FACS)-purified population of human CB CAFC content correlates well with colony-forming unit-spleen
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CD34 CD38 CD45RA Thy-1 cells that were additionally purified (CFU-S) content on day 12 and marrow repopulating capacity. 78,105
based on surface expression of the integrin α6 (CD49f) yielded 6.7- However, similarly to the LTC-IC, the CAFC assay does not measure
fold increased human donor chimerism at 20 weeks in NOD/SCID LT-HSCs. An advantage of the CAFC and LTC-IC assays is that the
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IL2Rγ (NSG) mice compared with injection with the identical estimate of stem/progenitor cell content is not confounded by the
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dose of CD34 CD38 CD45RA Thy-1 CD49f cells. Only the homing capacity of the cell population being tested. However,
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Thy-1 CD49f cells could be serially transplanted in this study, and competitive transplantation assays provide a more physiologically
the enrichment for LT-HSCs via limiting dilution analysis was esti- relevant measure of functional HSC content and allow quantification
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mated to be approximately one in 11 CD34 CD38 CD45RA Thy- of LT-HSC content as well as homing efficiency. 64,78,107
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1 CD49f cells. Further purification of this population of cells
using Rh123 dye demonstrated that single-cell transplantation of
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Thy-1 Rh123 CD49f cells yielded long-term, multilineage engraft- In Vivo Assays
ment in five of 18 transplanted recipients. Serial transplantation was
also successful in two of four secondary mice, suggesting that at least Colony-Forming Unit–Spleen Assay
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some of the Thy-1 Rh123 CD49f cells undergo self-renewal. Of The first reproducible in vivo assay for hematopoietic progenitor cells
note, because mice were transplanted via intrafemoral injection in (HPCs) was the CFU-S assay, which was developed by Till and
these studies, it remained unknown whether this panel of markers McCullough. 12,88 In this assay, BM cells are injected into lethally
equally identified human HSCs capable of homing properly to the irradiated mice, and macroscopic spleen colonies are measured from
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BM after intravenous injection. Nonetheless, these studies revealed one to three weeks after injection. These colonies represent short-
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that the addition of CD49f to the panel of human LT-HSC markers term repopulating cell and MPP activity but do not measure LT-HSC
provided an improved capability to isolate human HSCs at a level of content. 4,78
purity that was comparable to that applied to murine HSC isolation.
Recently, two novel cell surface markers for human HSCs Competitive Repopulation Assay
were identified, CD166 (activated leukocyte adhesion molecule) A significant advance in the study of hematopoiesis was the develop-
and protein tyrosine phosphatase-sigma (PTPσ). 101,102 Human ment of the competitive repopulating assay (Fig. 9.3). 78,108 In this assay,
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Lin CD34 CD38 CD49f CD166 cells engrafted in primary an unknown population of hematopoietic cells is transplanted via
and secondary NSG mice at a significantly higher level than intravenous injection into lethally irradiated syngeneic mice along
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Lin CD34 CD38 CD49f CD166 cells. Interestingly, CD166 is with a competing dose of host-derived BM cells. 78,109,110 This assay has
also expressed by BM osteoblasts and it was postulated that CD166 been refined over time such that it is typically performed using a limit-
mediated HSC maintenance in vivo via hemophilic interactions ing dilution method in which several cell doses (typically more than
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between CD166 expressed on HSCs and osteoblasts. Quarmyne three to five doses; n = 10 mice/dose level) of BM cells or purified
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et al reported that NSG mice transplanted with human CB HSCs (e.g., CD34 KSL cells) are injected into lethally irradiated mice
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Lin CD34 CD38 CD45RA PTPσ cells displayed 15-fold higher along with a fixed dose of host competitor BM cells, such that a fraction
human hematopoietic cell engraftment at 16 weeks compared of the recipient mice can be predicted to have nonengraftment. 78,111,112
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to mice transplanted with Lin CD34 CD38 CD45RA cells or This approach allows the application of Poisson statistical analysis to
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Lin CD34 CD38 CD45RA PTPσ cells. PTPσ was shown to provide an estimate of competitive repopulating units (CRUs) within
negatively regulate both murine and human HSC repopulation fol- the donor hematopoietic cell population. 78,111–113 An important feature
lowing transplantation, via inhibition of the RhoGTPase, RAC1. 102 of the CRU assay is the potential to estimate the frequency of LT-HSCs
in a given hematopoietic cell population. Donor cell engraftment that
Functional Characterization is detected within the first 8 to 12 weeks after transplantation, reflects
the contribution of ST-HSCs, which extinguish at or beyond 12 weeks
posttransplant. Therefore, the number of LT-HSCs cannot be con-
In Vitro Assays vincingly estimated until more than 12–20 weeks posttransplanta-
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tion. 78,114 Dykstra et al showed that competitive transplantation of
The colony-forming cell (CFC) assay does not measure HSC single, phenotypic HSCs results in stable donor cell engraftment in
content but rather committed myeloid progenitor cell content via lethally irradiated mice beyond 16 weeks, and retroviral marking of
a 14-day assay for colonies within methylcellulose media that is HSCs revealed that stable donor-derived hematopoiesis was not
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supplemented with specific growth factors. The CFC assay measures observed in recipient mice until six months posttransplant. 114
colony-forming unit-granulocyte/macrophage (CFU-GM), burst- A commonly used and rigorous approach to estimate the presence
forming unit-erythroid (BFU-E) and CFU-granulocyte/erythroid/ of LT-HSCs is the performance of secondary, tertiary, and quaternary
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macrophage/megakaryocyte (CFU-GEMM). The CFU-GEMM, or HSC transplants. This approach is based on the principle that a
CFU-mix colonies, represent a more immature progenitor cell popu- singular feature of primitive LT-HSCs is the capacity to serially
lation. B- and T-cell progenitor cell content can also be measured via reconstitute multilineage hematopoiesis in vivo without exhaus-
in vitro assays but requires specialized coculture conditions, which are tion. 78,116–118 In this method, whole BM is typically collected from
described elsewhere. 78,103,104 primary recipient mice and then injected, along with host competitor
The long-term culture-initiating cell (LTC-IC) assay is a 6-week BM cells, into lethally irradiated syngeneic mice. Donor cell repopu-
in vitro assay in which BM cells are cocultured with murine stromal lation is then measured at 12–20 weeks posttransplantation. Serial
cells for four weeks followed by replating of the entire culture system transplantation assays have the limitation of being potentially con-
into methylcellulose and additional two-week assay for colony forma- founded by variables such as homing efficiency of the donor
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tion. 78,105 The LTC-IC, unlike the CFC, measures a more immature cells. 78,119,120 Therefore, as pointed out by Purton and Scadden in
stem/progenitor cell population, although the results of the LTC-IC an excellent review of this subject, serial transplantation assays may
are inherently dependent and limited by technical variabilities in be better suited to studies of wild type hematopoietic cell populations
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stromal coculture experiments. Importantly, the LTC-IC popula- as opposed to mutant mice-derived hematopoietic cells, which may
tion lacks long-term repopulating cells because transplantation of have alterations in homing or engraftment mechanisms independent
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LTC-ICs into mice in a competitive transplantation assay does not of HSC content. Utilization of whole BM avoids issues regarding
result in any long-term reconstitution. 78,88,89 the fidelity of phenotypic markers of HSCs in mutant mice and is
The cobblestone area-forming cell (CAFC) assay also involves perhaps more broadly feasible than FACS-isolated HSC populations
coculture of HSCs with preestablished stromal cell monolayers and at some centers. 78,121–124 However, the use of purified HSCs avoids
relies on microscopic quantification of cobblestone-forming cells the potential confounding effects of accessory cells contained within
embedded underneath the stromal layer. 105,106 It has been shown that the BM graft on donor cell repopulation and allows for precise