Stromal cell-dependent ex vivo expansion of human cord blood progenitors and augmentation of transplantable stem cell activity

Abstract

Summary:In The remarkable proliferation and cell renewal processes in the human hematopoietic system are believed to be supported by a small population of hematopoietic stem cells. 1,2 A major challenge in stem cell research is the establishment of culture systems that facilitate in vitro maintenance and augmentation of stem cell activity. This is of great impor- tance not only for hematopoietic stem cell transplantation but also for gene therapy. It is also an important step towards cellular and molecular understanding of the regulatory mechanisms that mediate the commitment vs self-renewal decision of stem cells.In the last two decades a number of hematopoietic cytokines have been identified. Studies using recombinant hematopoietic cytokines have revealed that hematopoietic cytokines alone or in combination support the proliferation of hematopoietic progenitors and that it is possible to increase the number of progenitors in culture. 3 Recently, several groups of investigators have reported that combinations of early-acting cytokines support the maintenance or expansion of progenitors and even of transplantable stem cells in both murine 4-7 and human systems. [8][9][10][11] On the other hand, however, the importance of stromal cells in the maintenance and expansion of primitive progenitors including transplantable stem cells have been repeatedly demonstrated. 12-20 A number of stromal cell lines have been reported to support the proliferation of human primitive progenitors. MS-5 is one such cell line, derived from noninfected Dexter-type murine long-term marrow culture, which supports CFU-S maintenance 21 and acts synergistically with human growth factors to stimulate the formation of blast colonies and macroscopic colonies from CD34 + CD38 Ϫ primitive progenitors in short-term methylcellulose assays. 22 MS-5, alone 23 or in combination with SCF and G-CSF, 24,25 also supports the proliferation of primitive lymphohematopoietic progenitors that are capable of differentiation along both myeloerythroid and lymphoid lineages and their differentiation toward the B cell lineage. Furthermore, a recent study has demonstrated that, in the presence of multiple cytokines, MS-5 cells promote a net increase of LTC-IC through self-renewal divisions. 26 In the present study, we demonstrate synergistic effects of MS-5 cells and early-acting hematopoietic growth factors, FL and TPO on the expansion of human cord blood CD34 + cells, CD34 + CD38 Ϫ cells, CFU-C and CAFC. Using an SRC assay we also found that transplantable stem cell activity was slightly augmented or at least maintained in our culture system.

The Stromal Activity of Mesenchymal Stromal Cells

“…Neither IGFBP-1, IGFBP-2, nor IGFBP-3 had an effect on proliferation or maintenance of a primitive immunophenotype of HPC [39]. Many groups including our group have demonstrated that HSC can not be maintained in conditioned culture medium of feeder layer cells under non-contact conditions [11,47,60]. Although studies of other groups have suggested that primitive function of HPC might also be preserved in a transwell setting [40,61], there is evidence that soluble molecules alone are not enough to maintain long-term repopulating potential.…”

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells

“…[48][49][50][51][52][53] One component of the hematopoietic microenvironment, mesenchymal stem cells (MSCs), can be isolated as plastic adherent cells from a variety of fetal and adult tissues. [54][55][56][57] In addition to providing an alternative (co-culture) strategy for CB expansion, allogeneic MSC coadministration has been shown to promote engraftment of human CD34 þ cells in NOD/SCID mice and fetal sheep, [57][58][59][60][61] and MSCs have been shown to have clinical immunomodulatory activity that may impact on graft-versus-host disease in transplant patients. 59,[62][63][64][65][66][67][68][69] CB-MSC co-culture does not require the isolation of CD34 þ or CD133 þ cells before expansion, minimizing manipulation and loss of HPC.…”

Stromal cells from murine embryonic aorta–gonad–mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFCweek6 in extended long-term cultures

“…17,18 The production of CAFC week6 or LTC-initiating cells(IC) in ex vivo expansion cultures has been shown to be indicative for the presence and quantity of in vivo repopulating HSC as measured using the quantitative NOD/SCID xenotransplant model. 3,4,[19][20][21] However, behavior of the LTC-IC populations may only partly overlap with that of the HSC and show distinct characteristics under specific experimental conditions. 16,22 Screening a large number of stromal cell lines using this in vivo model is not practical.…”