Donor-to-donor variability in the expansion potential of human bone marrow cells is reduced by accessory cells but not by soluble growth factors.

Clinical trials assessing the utility of cultured hematopoietic cells for the support of patients receiving high-dose chemotherapy are beginning. Although many reports have described these cultures, little is known about the donor-to-donor variability that might be expected to occur in widespread use. Therefore, this study was undertaken to assess variables which might predict and reduce the donor-to-donor variability in cell expansion potential. CD34-enriched cell cultures, plated to contain 3000 CD34+lin- cells per well, exhibited a wide range of cell output (0.02 to 5.07 x 10(6)) with a high coefficient of variation (CV = 0.69, n = 52). The range in CFU-GM output was even greater (12 to 9455, CV = 0.90). Addition of preformed stroma had a significant positive effect, and resulted in narrower ranges of cell (0.19 to 8.27 x 10(6), CV = 0.41) and CFU-GM (218 to 17586, CV = 0.54) output. A wide range of stromal-dependency was exhibited by CD34-enriched cells from different donors, with stroma augmenting cell output by 1.2- to 14-fold (mean 3.5), and CFU-GM output by 1.7- to 24-fold (mean 6.5). In contrast, changes in the soluble growth factor combination affected cells from different donors in a similar fashion, thereby altering the mean level of performance without reducing donor-to-donor variability. Experiments were next performed to assess the relative contribution of CD34+lin- cells and stromal cells to culture variability by culturing CD34+lin- cells from three donors on preformed stroma from three donors in parallel. Variability in culture output was attributed to the CD34+lin- cell donor, whereas stroma from different autologous or allogeneic donors gave similar performance. Therefore, both expansion potential and stromal-dependency were inherent characteristics of CD34+lin- cells from different donors. Donor characteristics (i.e., sex, age, weight, and height) and flow cytometric assays (i.e., CD34+lin- cell purity, and CD38-, Thy-1+, and c-kit+ subsets thereof) were not well correlated with expansion potential. In contrast, many of the different biological characteristics (i.e., inoculum CFU-GM, cell and CFU-GM output, and stromal-dependency) were strongly correlated with each other. Mononuclear cell (MNC) cultures, which provide an accessory cell environment (including endogenous stroma) in which CD34+lin- cells grow, were compared with CD34-enriched cell cultures. MNC cultures (containing 3000 CD34+lin- cells) were found to give the greatest and most consistent cell (2.51 to 5.20 x 10(6), CV = 0.17) and CFU-GM (2618 to 14,745, CV = 0.46) output. These results have significant implications for the design of clinical trials of cultured hematopoietic cells, as well as for the understanding of diversity in human stem cell behavior. Furthermore, the results demonstrate the importance of a large sample size in scientific studies of primary human hematopoietic cell behavior.

Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production.

Perfusion cultures of human bone marrow mononuclear cells (BMMNC) provide a unique in vitro model of hematopoiesis, supporting growth of both accessory and hematopoietic elements. In this study, bioreactors were used to analyze the consumption and production of growth factors (GFs) in relation to each other and to the cells produced. The exogenously added GFs interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and erythropoietin (Epo) each exhibited different, but reproducible, consumption kinetics. Epo and IL-3 were consumed slowly for the first 5-7 days, and then the consumption rate of both increased. Epo consumption reached a plateau by day 10, whereas IL-3 consumption continued to increase. Consumption of SCF was similar to that of Epo, but began 2-3 days earlier. GM-CSF was consumed throughout the culture period in an accelerating manner. Consumption of SCF and Epo were related, because omission of Epo from the growth medium reduced SCF consumption by 53% and omission of SCF reduced Epo consumption by 82%. A reproducible relationship between cumulative GF consumption and total cell production was observed. Epo was most potent, with 5900 molecules consumed per cell produced, whereas 69,400 molecules of SCF were consumed per cell generated. More specifically, Epo consumption was correlated (r = 0.92 and 0.96) with the number of glycophorin A-positive (glyA+) cells produced, and the rate of Epo consumption varied with the progression of cells through the erythroid lineage. Consequently, measurement of GF consumption rates may be useful for quantifying the types of cells present in a culture. Endogenous GF production was also examined. G-CSF and MIP-1 alpha were present at high levels during the first 4 days but then declined rapidly. LIF first appeared in the second week and steadily increased thereafter. Omission of SCF from the medium allowed the detection of endogenous SCF production, and the kinetics was similar to that of LIF. IL-6 production was biphasic, with a peak and decline in week 1 and an increase during week 2. TGF-beta was below the level of detection in these cultures. The results suggest that perfusion supports accessory and hematopoietic elements which interact and therefore represent a partially functional tissue ex vivo. This system provides a useful model for studying relationships within GF networks and for elucidating the conditions that result in primitive cell expansion ex vivo.

Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow.

Growth of hematopoietic stem and progenitor cells found in the mononuclear cell (MNC) fraction of human cord blood and bone marrow was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Cord blood MNC were grown in suspension and on preestablished irradiated bone marrow stromal layers, whereas bone marrow MNC were used to initiate one-step long-term bone marrow cultures (LTBMC). Reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in all three types of long-term hematopoietic cultures (LTHC) studied. At various time points under low oxygen, progenitor cell numbers were as much as 12-fold, 3-fold, and 4-fold higher for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), respectively. In addition to these numerical increases, progenitor cells were maintained for 1-2 weeks longer under low oxygen conditions. Reduced oxygen tension also increased total cell numbers by as much as fivefold in cord blood suspension cultures, but this effect on total cell numbers was less pronounced in cultures containing a stromal layer. The rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination, was reduced under low oxygen. Finally, the beneficial effect of reduced oxygen was comparable to the effect of an irradiated stromal layer for maintaining cord blood progenitor cells in LTHC. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of both stromal and progenitor cells for a longer period of time in vitro.

Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid.

Flow cytometry has been used in recent years to study antigenic and physical changes accompanying hematopoietic cell differentiation. Such studies have provided the basis for rapid and objective assays that are potential alternatives to the colony assays currently in widespread use. In this report, erythropoiesis was examined in growth factor-supplemented perfused cultures of bone marrow mononuclear cells (BMMNC) using flow cytometric analysis of the transferrin receptor (CD71), glycophorin antigen expression occurred with time, Initially, fewer than 10% of the cells were (CD71++, but by day 8, 19-34% of the cells were CD71++gly A-. This was followed by the appearance of gly A on 10-60% of the CD71++ cells. After day 10, CD71 expression on many gly A+ cells decreased so that a population of CD71-gly A+ cells (11-54 accumulated by day 14. Each phenotype was sorted for morphologic identification and colony assay analysis. CD71++gly A- cells were 85% blasts, one-half of which were erythroblasts, and were significantly enriched for burst-forming units-erythroid (BFU-E). The time-varying number of CD71++gly A- cells in these cultures was found to correlate with the number of BF.78-0.97). Three-color analysis was next used to examine CD33 expression on BFU-E, and in fresh BM, most were found to be CD33-. During culture, however, the number of BFU-E recovered from CD33+ populations first increased and then decreased. Therefore, CD33 was not particularly useful for identifying BFU-E. In contrast, CFU-GM were mostly found to be in the CD71+CD33+ population throughout the culture period. When erythropoietin (Epo) was not added to these cultures, the percentage of gly A+ cells was reduced from 33 to 3.3%. Further, the omission of Epo caused an 80% decrease in the number of BFU-E and a corresponding 94% decrease in the number of CD71++gly A- cells, thereby maintaining the relationship between CD71++gly A- cells and BFU-E. Therefore, flow cytometric analysis was found to be useful in assessing erythroid development, and this approach may be used to develop flow cytometric assays for other populations of interest in hematopoietic cell cultures.

Direct contact between CD34+lin- cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production.

Perfused human bone marrow (BM) mononuclear cell (MNC) cultures result in a greater long-term culture-initiating cell (LTC-IC) output than parallel CD34+lin- cell cultures, even when CD34+lin- cells are placed on irradiated preformed stroma (IPFS). This difference has been attributed to accessory cell effects that are potentiated by medium perfusion. The present study investigated the relative contributions of direct contact- and soluble-mediated mechanisms of accessory cells in this culture system. CD34+lin- cells within (i.e., in contact with) the MNC accessory cell mixture generated greater LTC-IC output than CD34+lin- cells in contact with IPFS. Incubation of CD34+lin- cells with MNC conditioned medium (CM) resulted in partial restoration of MNC accessory activity, while CM from IPFS had no activity on LTC-IC output. Interestingly, the level of LTC-IC output supported by MNC CM was equivalent to that supported by direct contact with IPFS. CD34+lin- cells were then cultured in Transwell inserts either alone, with IPFS (direct contact), or with IPFS below the insert. Direct contact with IPFS significantly increased the output of cells, CFU-GM, and LTC-IC from CD34+lin- cells. IPFS below the insert also resulted in significantly increased cell and CFU-GM output, but did not significantly affect LTC-IC output. Further experiments using CM from CD34+lin- cells and IPFS cultures showed that LTC-IC supportive activity was present only when direct contact was allowed between CD34+lin- cells and IPFS. ELISA and RT-PCR experiments showed that contact did not induce changes in the levels of several known growth factors, including GM-CSF, IL-1beta, IL-3, IL-6, IL-11, LIF, KL, FL, Tpo, TGF-beta, and MIP-1alpha. These results indicate that direct contact between CD34+lin- cells and IPFS induces soluble activity, which specifically increases LTC-IC output from CD34+lin- cell cultures, providing evidence for a novel direct contact-mediated two-way mechanism of communication between primitive hematopoietic cells and stroma.

Tissue culture surface characteristics influence the expansion of human bone marrow cells.

Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application.

Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system.

Use of umbilical cord blood (CB) for stem cell transplantation has a number of advantages, but a major disadvantage is the relatively low cell number available. Ex vivo cell expansion has been proposed to overcome this limitation, and this study therefore evaluated the use of perfusion culture systems for CB cell expansion. CB was cryopreserved using standard methods and the thawed unpurified cells were used to initiate small-scale cultures supplemented with PIXY321,flt-3 ligand, and erythropoietin in serum-containing medium. Twelve days of culture resulted in the optimal output from most CB samples. Frequent medium exchange led to significant increases in cell (93%), CFU-GM (82%) and LTC-IC (350%) output as compared with unfed cultures. As the inoculum density was increased from 7.5 x 10(4) per cm2 to 6.0 x 10(5) per cm2, the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM output reached a plateau at 6.0 x 10(5) per cm2, whereas LTC-IC output continued to increase up to 1.2 x 10(6) per cm2. Because the increase in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 x 10(5) per cm2 for cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent stroma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-CSF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-beta, TNF-alpha, and MIP-1alpha) that were also found in bone marrow (BM) cultures, with the exception of IL-11 (found only in BM) and IL-3 (found in neither). Culture output was remarkably consistent from 10 CB samples (coefficient of variation 0.3) indicating that the procedure is robust and reproducible. Two commercial serum-free media were evaluated and found to support only approximately 25% of the culture output as compared with serum-containing medium. Implementation of optimal conditions in the clinical scale, automated cell production system (CPS) showed that the process scaled-up well, generating 1.7 x 10(7) CFU-GM (298-fold expansion) from 1.2 x 10(8) thawed viable nucleated CB cells (n = 3). The ability to generate >10(7) CFU-GM from <15 ml of CB within this closed, automated system without the need for extensive cell manipulations will enable clinical studies to test the safety and efficacy of expanded CB cells in the transplant setting.

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor.

Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized. Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations. We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges. Interleukin-3 (IL-3) and interleukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 2.4-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures. Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion. In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained. More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion. The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

Size of family of orientation and family size preference: a replication.

"Past studies have found a modest, positive association between size of family of orientation and preferred family size and fertility behavior in the family of procreation. While there is some consistency in the findings, contradictions are also noted. The present study replicates earlier studies dealing with preferred family size, and seeks to resolve some of the contradictions by utilizing multiple classification analysis to determine the simultaneous impact of parental family size, religion, happiness, life style stability, and birth order on family size preference." The data, collected in 1981-1982, concern 379 U.S. college students.

Review: tissue engineering: reconstitution of human hematopoiesis ex vivo.

The reconstruction of functioning human tissues ex vivo is becoming an important part of biotechnology. There are compelling scientific, clinical, and biotechnological reasons for fully or partially reconstituting human tissues such as skin, bone marrow, and liver ex vivo. In particular, bone marrow is a tissue of much importance, and there are significant societal and health benefits derived from a successfully constructed ex vivo hematopoietic system. In this article, we review the current status of this effort. The topics covered include the current understanding of the biology of human hematopoiesis, the motivation for reconstructing it ex vivo, the current state of ex vivo human hematopoietic cultures, the development of important metrics to judge culture performance, and an approach based on in vivo mimetics to accomplish this goal. We discuss some applications of functional ex vivo hematopoietic cultures and the biological and engineering challenges that face research in this area.

Cell adhesion in animal cell culture: physiological and fluid-mechanical implications.

We have reviewed the general forces through which cells interact with substrata in their first nonspecific contact. The complex, fast-emerging biology of specific cell adhesion and the structure of the extracellular matrix were reviewed in substantial detail, and the most updated conceptual model of biological cell adhesion was assembled from past efforts and new literature data. The chemistries of the various possible substrata for cell adhesion have been reviewed extensively in the past, and here only a brief summary was presented, with particular emphasis on the materials for traditional and porous microcarriers. The fascinating molecular and cellular implications of cell adhesion were reviewed in detail to establish that cell adhesion and the extracellular matrix provide more than structural support for the cells and their assemblies, and that in fact they constitute fundamental regulators of cell function, metabolism, and differentiation. We reviewed the fluid-mechanical mechanisms of cell damage in microcarrier systems and provided experimental evidence for the importance of the cell-adhesion quality in the ability of cells to withstand fluid forces in bioreactors. We provided evidence that the interplay of cell adhesion and fluid forces is likely to produce cell responses more complex than that of simple life and death, and we suggested that such responses are awaiting investigation and exploration for new applications and culturing possibilities. We also reviewed the experimental evidence on the importance of cell adhesion in cell and microcarrier aggregation and discussed the implications of such aggregation on the culturing environment and the operation of bioreactors. Finally, we discussed the possible implications of cell adhesion as it relates to the developing field of tissue engineering, using the example of bone marrow culture, which involves a large variety of cells and constitutes one of the most complex cell culture systems.

Unilineage model of hematopoiesis predicts self-renewal of stem and progenitor cells based on ex vivo growth data.

Stem cell models are used to describe the function of several tissues. We present unilineage kinetic description of stem cell models and their application to the analysis of ex vivo hematopoietic cell expansion data. This model has the capability to simulate the total cell number and the number of cells at each stage of differentiation over time as a function of the stem cell self-renewal probability, the growth rate of each subpopulation, and the mature cell death rate. The model predicts experimental observations in perfusion-based hematopoietic bioreactor systems. To obtain net cell expansion ex vivo, the model simulations show that the stem cell self-renewal probability must exceed one-half, thus resulting in net expansion of the stem cell population. Experimental data on long-term culture-initiating cells (LTC-IC) confirm this prediction and the probability of self-renewal is estimated to be 0.62 to 0.73. This self-renewal probability, along with the death rate, define a relationship in which the apparent overall growth rate is less than the compartmental growth rate. Finally, the model predicts that cells beyond the stem cell stage of differentiation must self-renew to achieve the level of expansion within the time frame observed in experimental systems. (c) 1996 John Wiley & Sons, Inc.

Frequent harvesting from perfused bone marrow cultures results in increased overall cell and progenitor expansion.

The establishment of prolific long-term human bone marrow cultures has led to the development of hematopoietic bioreactor systems. A single batch expansion of bone marrow mononuclear cell populations leads to a 10- to 30-fold increase in total cell number and in the number of colony forming units-granulocyte/macrophage (CFU-GMs), and a four- to tenfold increase in the number of long-term culture initiating cells (LTC-ICs). In principle, unlimited expansion of cells should be attainable from a pool of stem cells if all the necessary requirements leading to stem cell maintenance and division are met. In this article, we take the first step toward the identification of factors that limit single batch expansion of ex vivo bone marrow cells in perfusion-based bioreactor systems. One possible constraint is the size of the growth surface area required. This constraint can be overcome by harvesting half the cell population periodically. We found that harvesting cells every 3 to 4 days, beginning on day 11 of culture, led to an extended growth period. Overall calculated cell expansion exceeded 100-fold and the CFU-GM expansion exceeded 30-fold over a 27-day period. These calculated values are based on growth that could be obtained from the harvested cell population. Growth of the adherent cell layer was stable, whereas the nonadherent cell population diminished with increasing number of passages. These results show that the bioreactor protocols published to date are suboptimal for long-term cultivation, and that further definition and refinement is likely to lead to even greater expansion of hematopoietic cell populations obtained from bone marrow. More importantly, these results show that the LTC-IC measured during the single pass expansion do have further expansion potential that can be realized by frequent harvesting. Finally, the present culture conditions provide a basis for an assay system for the identifications provide a basis for an assay system for the identification of the factors that determine the long-term maintenance and replication of human stem cells ex vivo.

Importance of parenchymal:stromal cell ratio for the ex vivo reconstitution of human hematopoiesis.

Many new developments in tissue engineering rely on the culture of primary tissues which is composed of parenchymal and mesenchymal (stromal) cell populations. Because stroma regulates parenchymal function, the parenchymal:stromal cell (P:S) ratio will likely influence culture behavior. To investigate parenchymal-stromal cell interactions, the P:S ratio was systematically varied in a human bone marrow (BM) culture system, measuring the output of mature cells, immature progenitors (colony forming units-granulocyte/macrophage [CFU-GM]), and primitive stem cells (long-term culture-initiating cells [LTC-IC]). When parenchymal CD34-enriched cells were grown without stroma, cell and CFU-GM output increased linearly as inoculum density was increased, resulting in constant cell and CFU-GM expansion ratios. On irradiated preformed stroma (IPFS), culture output was significantly higher and less dependent on CD34-enriched cell inoculum density, resulting in greater expansion ratios at lower inoculum densities. The number of IPFS cells required to support CD34-enriched cells was independent of the CD34-enriched cell number, suggesting that IPFS did not provide discrete niches, but instead acted through soluble signals. Experiments using conditioned medium (CM) from IPFS confirmed the presence of soluble signals, but CM did not completely substitute for direct contact between CD34-enriched cells and IPFS. Because of known differences between IPFS and stroma growing within BM mononuclear cell (MNC) cultures, experiments were next performed using mixtures of CD34-enriched and CD34-depleted fractions of MNC. When inoculated with a fixed CD34+lin- cell number, culture output was optimal near the P:S ratio of the unmanipulated MNC sample and declined as CD34- cell number was increased or decreased. In cultures inoculated with a fixed total cell number, CFU-GM output increased as CD34+lin- cell number was increased, whereas LTC-IC output reached a plateau. These data suggest that a limited number of LTC-IC supportive niches were present in MNC stroma, whereas IPFS lacks these niches and acts predominantly through a less potent soluble mechanism. These studies underscore the importance of parenchymal-stromal cell interactions in the ex vivo reconstitution of tissue function and offer insight into the nature of these interactions in the human BM culture system.

Quantitative long-term culture-initiating cell assays require accessory cell depletion that can be achieved by CD34-enrichment or 5-fluorouracil exposure.

Characterization of hematopoietic cells and measurement of their proliferative potential is critical in many research and clinical applications. Because in vivo assay of human cells is not possible and xenogeneic assays are not yet routine, in vitro assays such as the long-term culture-initiating cell (LTC-IC) assay have been widely adopted. This study investigated LTC-IC assay linearity and reproducibility and resulting implications with respect to quantitation of primitive cell expansion. Measurement of secondary colony-forming cells (2 degrees CFCs) from 5-week cultures of bone marrow (BM) mononuclear cells (MNCs) showed that 2 degrees CFC frequency varied with assay plating density in a nonlinear fashion. The measured 2 degrees CFC frequency increased from 4.6 to 63.8 (per 10(5) MNCs) as assay plating density was decreased from 5 x 10(5) to 2 x 10(4) MNCs per well (P < 10(-6), n = 37). In contrast, assay of CD34-enriched cells was linear within the range studied. Assays of cells obtained from expansion cultures initiated with either MNCs or CD34-enriched cells were also nonlinear. Consequently, calculated 2 degrees CFC expansion ratios were ambiguous and dependent on the assay plating densities used. Limiting dilution analysis (LDA) results were also nonlinear, with LTC-IC frequency increasing from 8. 2 to 22.4 per 10(5) MNCs (P < 10(-4), n = 100) as assay plating densities were decreased. Despite the nonlinearity, 2 degrees CFC and LTC-IC assay results were consistent and reproducible over time with different samples and techniques and gave a semiquantitative indication of relative primitive cell frequency. Although CD34-enriched cells gave linear assay output, purification of cells for every assay is impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was explored for improving assay linearity. Incubation of MNCs in 250 microg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in a cell population that gave linear 2 degrees CFC readout. The 5-FU-resistant LTC-ICs accounted for 49% of the total LTC-IC population, adding the potential benefit of restricting assay measurement to more primitive noncycling LTC-ICs. Consequently, similar linear assay results can be obtained with either the bulk 2 degrees CFC or LDA LTC-IC methods after 5-FU, but multiple plating densities are nevertheless still required in both methods due to the greater than 100-fold range in primitive cell frequency present in normal human donor BM.

Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells.

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC(8) (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC(5) in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC(8). The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC(5) expansion has now been extended to LTC-IC(8) expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.

Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures.

There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU-GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8-fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.

flt-3 ligand is more potent than c-kit ligand for the synergistic stimulation of ex vivo hematopoietic cell expansion.

The c-kit and flt-3 tyrosine kinase receptors are expressed on primitive hematopoietic cells, and ligands for both receptors have been cloned. In this study, the effects of c-kit ligand (KL) and flt-3 ligand (FL) were compared in the presence of IL-3, GM-CSF, and erythropoietin (3/GM/EPO), using frequent medium exchange cultures of human bone marrow mononuclear cells (BMMNC) and CD34-enriched cells. In MNC cultures, KL increased cell output by 1.7-fold (p < 10(-4), n = 13) and CFU-GM output by 2.4-fold (p < 10(-3)) as compared with control cultures containing only 3/GM/EPO. Analogously, FL increased cell output by 1.3-fold (p < 10(-3)) and CFU-GM output by 4.4-fold (p < 10(-6)). Therefore, FL was more potent on CFU-GM output than KL, but neither altered the lineage composition (granulocyte, monocyte, macrophage) of the colonies produced. Direct addition of KL or FL to colony assays resulted in only a 1.2-fold increase in CFU-GM outgrowth, suggesting that the effects on increased CFU-GM output were at the preprogenitor stage. In CD34-enriched cell cultures, the effects of KL and FL on CFU-GM output were similar (9-fold above control). Nevertheless, MNC cultures (containing an equivalent number of CD34+lin- cells) always generated more cells (2-fold to 4-fold) and CFU-GM (3-fold to 6-fold) than did parallel cultures of CD34-enriched cells. The greater effect of FL (over KL) in MNC cultures was probably due to synergy with endogenously produced growth factors that were absent in CD34-enriched cell cultures. FL-containing cultures (+/-KL) generated cells that formed larger colonies, and these cells had more proliferative potential on replating into secondary and tertiary cultures. Furthermore, FL increased the output of LTC-IC by 2.1-fold (p < 0.01) and CD34+lin- cells by 6-fold (p < 0.05) as compared with 3/GM/EPO cultures. In contrast, KL did not affect the output of LTC-IC and only slightly increased CD34+lin- cell output (by 1.4-fold). Erythrocytes were increased by KL (2.8-fold) and decreased by FL (0.6-fold), whereas granulocytes and monocytes were increased by both KL (1.4-fold) and FL (2.0-fold). When used together, KL and FL were completely additive with respect to cell, CFU-GM, and LTC-IC output, as well as lineage composition. The results indicate that FL is a more potent synergistic growth factor than KL for MNC expansion and that KL and FL act in an independent, direct, additive manner.