Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.

Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch B, Dick JE

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.

PMID: 9254660, UI: 97402493

Blood 1997 Jul 15;90(2):641-650

Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.

Gan OI, Murdoch B, Larochelle A, Dick JE

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

PMID: 9226164, UI: 97369723
Gene Ther 1997 Jul;4(7):744-749 

A rapid screening procedure for the identification of high-titer retrovirus packaging clones.

Murdoch B, Pereira DS, Wu X, Dick JE, Ellis J

We have developed a viral RNA (vRNA) dot blot assay for rapid identification of high-titer retrovirus vector production by packaging cell clones. The procedure employs Trizol LS reagent to purify vRNA from packaging cell supernatants, a sensitive dot blot assay, and Phosphorlmager technology to quantify packaged viral genomes in 2 days. Experiments performed on viral supernatants of known biological titer demonstrated that the vRNA dot blot assay was extremely sensitive and that dot intensity correlated directly with viral titer. It is often necessary to analyze approximately 100 virus producing cell clones, making this method useful as a rapid screen to identify the highest virus producing clones. The vRNA dot blot assay consistently identified a subset of candidate high-titer producer cell clones. In three independent screens the supernatant with the highest biological titer was produced by one of the previously defined candidate high-titer producer clones. Our procedure greatly facilitates virus titration by: (1) rapidly eliminating the vast majority of low-titer producer cell clones; (2) accurately identifying the subset of candidate high-titer producer clones for further biological titration and assessment of the proviral genomic structure; and (3) reducing laborious tissue culture manipulations to a minimum. Furthermore, the reliance of this method on molecular detection makes it ideally suited for the isolation of high-titer clones lacking a drug selection marker.

PMID: 9282176, UI: 97427056 

Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.

Wang JC, Doedens M, Dick JE

We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

PMID: 9166828, UI: 97309413 

Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.

Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. These SCID-repopulating cells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34(+) CD38(-) cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400, 000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34(+) CD38(-) cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells during in vitro culture.

PMID: 9144235, UI: 97289764

Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy.

Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE

The development of stem-cell gene therapy is hindered by the absence of repopulation assays for primitive human hematopoietic cells. Current methods of gene transfer rely on in vitro colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, as well as inference from other mammalian species. We have identified a novel human hematopoietic cell, the SCID-repopulating cell (SRC), a cell more primitive than most LTC-ICs and CFCs. The SRC, exclusively present in the CD4+CD8- fraction, is capable of multilineage repopulation of the bone marrow of nonobese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). SRCs were rarely transduced with retroviruses, distinguishing them from most CFCs and LTC-ICs. This observation is consistent with the low level of gene marking seen in human gene therapy trials. An SRC assay may aid in the characterization of hematopoiesis, as well as the improvement of transduction methods.

PMID: 8946831, UI: 97102404