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Stem Cells, Vol. 16, No. 2, 127-135, March 1998
© 1998 AlphaMed Press

Kinetics of Hematopoiesis in Dexter-Type Long-Term Cultures Established from Human Umbilical Cord Blood Cells

Hector Mayania, Margarita Gutiérrez-Rodrígueza, Laura Espinozaa, Edith López-Chalinib, Alejandra Huerta-Zepedaa, Eugenia Floresa, Elizabeth Sánchez-Vallea, Fernando Luna-Bautistaa, Ignacio Valenciaa, Octavio T. Ramírezb

a Oncological Research Unit, Oncology Hospital, National Medical Center, IMSS, Mexico City, Mexico;
b Bioengineering Department, Institute of Biotechnology, UNAM, Cuernavaca, Mexico

Key Words. Bone marrow • Cord blood • Dexter culture • Hematopoiesis • Progenitors • Stroma

Dr. Hector Mayani, Oncological Research Unit, Oncology Hospital, National Medical Center, Av. Cuauhtemoc 330, Mexico, D.F. 06720.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we have established Dexter-type long-term cultures (D-LTC) from human umbilical cord blood (UCB) and followed the kinetics of different hematopoietic progenitor cells (HPCs)—including multipotent (colony forming unit [CFU]- Mixture), erythroid (CFU-erythroid, BFU-E), and myeloid (CFU-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage) progenitors as well as of morphologically recognizable erythroid, myeloid and lymphoid cells—during a nine-week culture period. D-LTC were also established from adult bone marrow (BM) as controls. On day 0, both UCB and BM showed similar total numbers of HPCs (about 310/105 cells), however, UCB showed a higher proportion of primitive HPCs (i.e., CFU-Mixture, CFU-granulocyte/macrophage and BFU-E). A poor adherent cell layer, consisting almost exclusively of macrophages, was developed in UCB D-LTC and this correlated with a continuous decline in HPC numbers throughout the culture period. In contrast, adherent cell numbers in BM D-LTC, including fibroblasts and macrophages, were two- to fourfold higher than in UCB cultures, and the numbers of HPCs were also significantly higher, reaching plateau levels between weeks 6 and 9. In both types of cultures, erythroid and multipotent progenitors declined relatively fast, reaching undetectable levels after five weeks of culture. Myeloid progenitors, on the other hand, were sustained longer (always at higher levels in BM cultures) and were still detected by week 9. Among myeloid progenitors, a shift towards the predominance of macrophage HPCs was observed, both in UCB and BM D-LTC, and this correlated with an increase in the proportion of mature monocytes and macrophages. Taken together, our results indicate that myeloid progenitor cell growth is deficient in UCB D-LTC and suggest that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures regardless of the number and types of adherent cells present; this suggests that the stroma developed in D-LTC is not sufficient for the proliferation of these progenitor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Umbilical cord blood (UCB) has been recognized as a major source of hematopoietic progenitor cells (HPCs) [1, 2]. Indeed, it has been shown that UCB contains a proportion of HPC that is at least comparable with that of bone marrow (BM) [3, 4] and this has led to the clinical use of UCB as an alternative to BM for hematopoietic cell transplantation [5]. By culturing UCB CD34+ cell subpopulations, both in semisolid and liquid cultures, it has been possible to characterize their proliferation and differentiation patterns. Furthermore, liquid cultures supplemented with different hematopoietic cytokine combinations have been used for the ex vivo expansion of both primitive and mature UCB HPCs [6-10], a procedure of potential clinical relevance [11, 12]. Interestingly, major functional differences between UCB and BM HPCs have been described, both in vitro and in vivo, indicating that UCB-derived HPCs possess a higher proliferation potential than their marrow counterparts [3, 13-16].

Much of our current knowledge on the in vitro proliferation and differentiation of HPCs—particularly those from BM—has derived from studies using Dexter-type long-term cultures (D-LTC) [17]. In this experimental system hematopoietic cell growth occurs in close association with an adherent cell layer formed by marrow stromal elements, which are derived from the same marrow sample. Under these culture conditions, hematopoiesis can be sustained for several weeks, or even months, without the addition of exogenous cytokines, since marrow stromal cells produce both extracellular matrix proteins and a variety of cytokines necessary for the development of hematopoietic cells [18-20].

To date, however, limited information exists on the behavior of UCB cells in standard D-LTC, that is, in the absence of a preformed allogeneic marrow stroma and without the addition of recombinant cytokines. The studies reported to date have only partially analyzed UCB hematopoiesis in this experimental system since they have focused exclusively on total myeloid progenitors present in the nonadherent fraction of the culture [3, 4]. Thus, the major goal of the present study was to thoroughly characterize UCB hematopoiesis in D-LTC by following the growth kinetics of nonadherent and adherent progenitor cells (including multipotent and the different types of myeloid and erythroid progenitors), as well as morphologically recognizable cells, throughout a nine-week culture period. For comparison, D-LTC from normal adult BM were simultaneously analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Collection
UCB cells, collected according to institutional guidelines, were obtained from 10 normal full-term deliveries from the "1° de Octubre" and "20 de Noviembre" Hospitals (Mexico City, Mexico). Eight normal BM samples were obtained, one of them from the sternum of a patient undergoing cardiac surgery at the Cardiology Hospital, National Medical Center (Mexico City, Mexico) and the other seven from the iliac crest of BM transplant donors, at the "Bernardo Sepulveda" Hospital, National Medical Center. These procedures have been approved by the ethical committee of the National Medical Center.

Cell Processing
Buffy coat cells, both from UCB and BM, were obtained by centrifugation (400 g for seven min) and resuspended in Iscove's modified Dulbecco's medium (IMDM) supplemented with 2% fetal bovine serum ([FBS]; StemCell Technologies, Inc.; Vancouver, BC, Canada). Total numbers of nucleated and viable cells were determined with a hemocytometer, using Turck's solution and trypan blue stain, respectively.

Long-Term Cultures
D-LTC were established based on the method described by Eaves et al. [21] and Mayani et al. [22]. Buffy coat cells were resuspended in LTC medium (StemCell Technologies, Inc.) at a final concentration of 3 x 106 cells per ml. The LTC medium composition is as follows: alpha medium supplemented with 12.5% horse serum, 12.5% FBS, 0.2 mM inositol, 20 µM folic acid, 10–4 M 2-mercaptoethanol, 2 mM L-glutamine and freshly dissolved hydrocortisone to yield a final concentration of 10–6 M. The cell suspension was loaded into 24-well plates (1 ml/well) and incubated at 37°C in an atmosphere of 5% CO2 in air. After three days, cultures were transferred to a different incubator and maintained at 33°C. Four days later, seven days after initiation of the culture, half of the supernatant and nonadherent cells were removed from the wells and replaced with fresh culture medium. The cultures were processed in this manner at weekly intervals, and at least two wells per UCB or BM culture were evaluated at each time point. The nonadherent cells, obtained weekly during the medium change, were counted, morphologically analyzed and assayed for hematopoietic progenitors. The well-to-well variability observed in progenitor cell frequency, both in UCB and BM cultures, was <12%. At weeks 3, 5, 7 and 9 one of a number of parallel cultures was sacrificed for evaluation of the adherent cells, which were detached with a cell scraper after trypsinization (i.e., 0.25% trypsin containing 0.1 mM EDTA was added and the cultures were incubated at 37°C for 10 min; the action of trypsin was stopped by adding one-half volume of FBS). The cells were then resuspended in IMDM with 2% FBS and processed in the same way as the nonadherent cells.

Hematopoietic Colony Assays
HPCs were assayed in methylcellulose-based semisolid cultures (StemCell Technologies, Inc.). The culture medium consisted of 0.9% methylcellulose, 30% FBS, 1% bovine serum albumin, 10–4 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant human (rHu) stem cell factor, 10 ng/ml rHu interleukin 3, 10 ng/ml rHuGM-CSF and 3 U/ml rHu erythropoietin. Buffy coat cells, both from UCB and BM, were plated at a final concentration of 5 x 104 cells/ml and the cultures were incubated at 37°C in an atmosphere of 5% CO2 in air. Nonadherent and adherent cells from D-LTC were also cultured in this manner; however, the plating cell concentration varied from 5 x 104 to 1 x 104, depending on the cell number recovered from the cultures. After 14-17 days of culture, colonies were scored in the same dish using an inverted microscope. Hematopoietic colonies were classified as previously described [22]: colony forming unit-Mixture (CFU-MIX), colonies containing both erythroid and myeloid cells; CFU-erythroid (CFU-E), erythroid clusters of 20-50 hemoglobinized cells; BFU-E, erythroid colonies of more than 50 hemoglobinized cells grouped in one or several clusters. Myeloid colonies comprised the identifiable subpopulations of pure granulocytic colonies (CFU-granulocyte [CFU-G]), pure macrophagic colonies (CFU-macrophage [CFU-M]), and colonies containing both granulocytes and macrophages (CFU-granulocyte/macrophage [CFU-GM]).

Morphological Studies
Cells obtained from UCB and BM, as well as from the nonadherent fraction of D-LTC, were examined on slide preparations stained with Wright-Giemsa stain. Approximately 500 cells per slide were scored.

Statistics
Statistical analysis was performed by using Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPC Content in UCB and BM
HPC content, both in UCB and BM samples, was determined by colony assays. A mean total of 303 colony forming cells (CFC) per 105 nucleated cells (NC) was observed in UCB samples. Forty-three percent of them (132/105 NC) corresponded to myeloid progenitors; 50% (153/105 NC) were erythroid progenitors, and 6% (18/105 NC) corresponded to multipotent progenitors ( Table 1). A mean total of 321 CFC/105 NC was observed in BM samples. Fifty-one percent of them (160/105 NC) corresponded to myeloid progenitors, 48% (155/105 NC) to erythroid progenitors and only 1% (5/105 NC) to multipotent progenitor cells. No statistical difference was found for myeloid or erythroid progenitors when comparing their numbers in UCB and BM; in contrast, the numbers of multipotent progenitors were significantly higher in UCB than in BM ( Table 1).


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  		Table 1. Number of HPCs in UCB and BM
 
Among myeloid progenitors present in UCB, CFU-G corresponded to 33%, CFU-M corresponded to 40% and CFU-GM corresponded to 29%. In contrast, in BM, CFU-G corresponded to 62%, CFU-M corresponded to 32% and CFU-GM corresponded to 9% ( Table 2). Thus, a higher proportion of immature, bipotent progenitors was observed in UCB than in BM. In terms of erythroid progenitors, early progenitor cells (BFU-E) were also present at higher frequencies in UCB than in BM (94% versus 62%; p < 0.05; Table 3).


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  		Table 2. Relative proportion of myeloid progenitors in D-LTC throughout a nine-week culture period
 

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  		Table 3. Relative proportion of erythroid progenitors in D-LTC throughout a nine-week culture period
 
Kinetics of Nucleated Cells
BM nonadherent and adherent cells showed similar kinetics in D-LTC to those reported previously [21-23]. There was a rapid decline in nonadherent cells during the first few weeks of culture; afterwards, the rate of decline was considerably reduced and a plateau was observed between weeks 6-9 ( Fig. 1). Adherent cells were observed from week 1, and at weeks 5-9 their numbers were significantly higher (two- to sevenfold) than the numbers of nonadherent cells ( Fig. 1). According to morphological criteria, fibroblasts, macrophages and adipocytes were identified within the adherent cell layers developed in BM D-LTC (not shown).



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  		Figure 1. Absolute numbers of nucleated cells in human D-LTC established from UCB (left panel) and adult BM (right panel). Points represent mean ± SD of 10 experiments for UCB and eight experiments for BM (six UCB samples and five BM samples for week 9). NA = nonadherent cells; AD = adherent cells. No statistical difference was observed between NA cells in UCB and BM cultures. At weeks 3, 5 and 7 adherent cells in BM cultures were significantly higher (p < 0.05) than in UCB cultures; however, no statistical difference was observed at week 9.

 
In D-LTC initiated with UCB cells, the levels of nonadherent cells throughout most of the culture period were similar to those observed in BM D-LTC ( Fig. 1). However, in contrast to BM cultures, the numbers of adherent cells in UCB D-LTC were significantly reduced. In fact, at week 3, there were more nonadherent than adherent cells, and by week 5 their levels were about the same ( Fig. 1). Preliminary analysis indicates that adherent cell layers in UCB D-LTC consisted almost exclusively of macrophages; very few fibroblast-like cells and no adipocytes were observed (Gutiérrez-Rodríguez and Mayani, study in progress). At weeks 3, 5 and 7 adherent cell numbers were significantly higher (two- to fourfold; p < 0.05) in BM D-LTC than in their UCB counterparts. Only at week 9 the higher number of adherent cells in BM D-LTC was not statistically significant.

Kinetics of Myeloid Progenitors
In BM D-LTC, nonadherent myeloid progenitors showed a continuous decline during the first six weeks of culture and afterwards their levels remained constant ( Fig. 2). At weeks 3, 5, 7 and 9 adherent myeloid progenitors were evaluated and their levels were similar to those in the nonadherent fraction.



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  		Figure 2. Absolute numbers of myeloid progenitor cells (CFU-G + CFU-M + CFU-GM) in human D-LTC established from UCB (left panel) and adult BM (right panel). Points represent mean ± SD of 10 experiments for UCB and eight experiments for BM (six UCB samples and five BM samples for week 9). NA = nonadherent progenitor cells; AD = adherent progenitor cells. A statistical difference (p < 0.05) was observed at weeks 8 and 9 between NA myeloid progenitors in UCB and BM cultures. At all time points analyzed, the numbers of adherent myeloid progenitors in BM cultures were significantly higher than in UCB cultures (p < 0.05).

 
In UCB D-LTC, nonadherent myeloid progenitors showed a continuous decline throughout the entire culture period ( Fig. 2). In contrast to BM cultures, the levels of adherent myeloid progenitors in UCB D-LTC were significantly lower than in the nonadherent fraction and they also declined, reaching undetectable levels by week 9 ( Fig. 2). When both nonadherent and adherent myeloid progenitors were taken together, their total numbers were higher in BM D-LTC than in UCB cultures; this was particularly significant at weeks 3-9 ( Table 4). Interestingly, the relative proportion of myeloid progenitors throughout the culture period showed a shift towards the predominance of CFU-M, which corresponded to 72%-100% of the myeloid progenitors detected at week 9 ( Table 2). This was observed in BM D-LTC as well as in UCB cultures.


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  		Table 4. Total number of HPC (nonadherent + adherent) in D-LTC
 
Kinetics of Erythroid Progenitors
A continuous decline in the numbers of erythroid progenitors was observed in BM D-LTC; indeed, at week 7, these progenitors were practically absent both in the nonadherent and adherent fractions of the cultures ( Fig. 3). At weeks 3, 5 and 7 erythroid progenitors in the adherent fraction showed similar numbers to those observed in the nonadherent fraction.



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  		Figure 3. Absolute numbers of erythroid progenitor cells (CFU-E + BFU-E) in human D-LTC established from UCB (left panel) and adult BM (right panel). Points represent mean ± SD of 10 experiments for UCB and eight experiments for BM (six UCB samples and five BM samples for week 9). NA = nonadherent progenitor cells; AD = adherent progenitor cells. A statistical difference (p < 0.05) was observed only at week 8 between NA erythroid progenitors in UCB and BM cultures.

 
In UCB D-LTC, nonadherent erythroid progenitors also showed a continuous decline, although this was not as pronounced as in BM cultures ( Fig. 3). In fact, at week 8, these progenitors were still detected in UCB D-LTC, although at very low levels. Adherent erythroid progenitors showed lower numbers than their nonadherent counterparts and could not be detected after seven weeks of culture ( Fig. 3). When both nonadherent and adherent erythroid progenitors were taken together, no significant differences were observed between BM and UCB cultures ( Table 4). At all time points, the majority of the erythroid progenitors observed in UCB and BM D-LTC consisted of BFU-E, although a significant increase in the relative proportion of CFU-E was observed in UCB cultures throughout five weeks of culture ( Table 3).

Kinetics of Multipotent Progenitors
Among the different types of progenitor cells detected in D-LTC throughout the culture period, multipotent progenitors (CFU-MIX) showed the lowest levels and the fastest disappearance ( Fig. 4). In BM cultures, CFU-MIX were detected during the first five weeks, and similar numbers were observed both in the nonadherent and adherent fractions ( Fig. 4). In UCB cultures, a continuous decline in nonadherent CFU-MIX numbers was observed, reaching undetectable levels by week 6 ( Fig. 4). Adherent CFU-MIX were detected only at week 3 and at very low levels. On weeks 3 and 5 the total numbers of CFU-MIX (nonadherent plus adherent) were similar in UCB and in BM cultures. It was only on day 0 that significantly higher numbers of CFU-MIX were observed in UCB D-LTC ( Table 4).



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  		Figure 4. Absolute numbers of multipotent progenitor cells (CFU-MIX) in human D-LTC established from UCB (left panel) and adult BM (right panel). Points represent mean ± SD of 10 experiments for UCB and eight experiments for BM (six UCB samples and five BM samples for week 9). NA = nonadherent progenitor cells; AD = adherent progenitor cells. A statistical difference (p < 0.05) was observed at week 3 between adherent progenitors in UCB and BM cultures.

 
Kinetics of Morphologically Recognizable Cells
The kinetics of the morphologically recognizable cell lineages present in the supernatant of both UCB and BM D-LTC were analyzed throughout the nine-week culture period ( Fig. 5). In BM cultures, granulocytic and monocytic cells showed opposite kinetics. The former were the most numerous (66% of all nucleated cells) during the first week of culture and then declined drastically, comprising 14% of all nucleated cells by week 9. The latter, on the other hand, corresponded to 12% of the cells at week 0, and by week 9 their levels reached 78%. Lymphoid cells (16% of all nucleated cells at week 0) showed only a slight decline, whereas erythroblasts were always the less abundant cell population (<10% of all nucleated cells) ( Fig. 5). In contrast to BM cultures, in UCB D-LTC granulocytic cell levels were always between 40%-60% of all nucleated cells, whereas monocytes increased their levels from 4% on day 0 to 35%-50% by weeks 8-9; their levels, however, were significantly lower (p < 0.05) than in BM cultures at weeks 8-9. There was a decrease in the relative proportion of lymphoid cells, which comprised 34%-38% of all nucleated cells during the first week of culture (significantly higher [p < 0.05] than in BM cultures) and 8%-10% of all nucleated cells between weeks 8 and 9. Finally, erythroblasts showed a similar kinetics to the one observed in BM cultures, comprising less than 5% of the nucleated cells at all time points ( Fig. 5).



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  		Figure 5. Relative proportion of lymphoid cells (L), granulocytes (G), monocytes/macrophages (M) and erythroblasts (E) in the nonadherent fraction of human D-LTC established from UCB (left panel) and adult BM (right panel). Points represent means of 10 experiments for UCB and eight experiments for BM (six UCB samples and five BM samples for week 9).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Much of our current knowledge on the in vitro biology of UCB HPCs has derived from studies in which these cells are cultured on a preformed marrow stromal cell layer [3, 4] or in stroma-free liquid cultures supplemented with exogenous recombinant cytokines [6-10]. However, only limited information exists on the in vitro behavior of UCB HPCs in standard D-LTC, that is, liquid cultures established in the absence of a preformed allogeneic stroma and without the addition of recombinant cytokines. Indeed, previous studies looking at the growth of hematopoietic progenitors in UCB D-LTC have focused exclusively on myeloid progenitors present in the nonadherent fraction [3, 4]. Thus, in the present study we have characterized the growth kinetics of UCB hematopoietic progenitor and mature cells in D-LTC, throughout a nine-week culture period. D-LTC were also established from BM, as controls.

On day 0, similar numbers of total HPC were observed both in UCB and BM samples. However, we observed a higher proportion of primitive progenitors in UCB. Indeed, multipotent (CFU-MIX), myeloid bipotent (CFU-GM), as well as early erythroid (BFU-E) progenitor cell numbers were higher in UCB than in BM (3.6-fold, 2.7-fold and 1.5-fold, respectively). These results confirm and expand previous studies showing higher levels of CFU-MIX and BFU-E in UCB than in BM [3, 14]. In terms of erythroid progenitors, the relative proportion of CFU-E and BFU-E, both in UCB and BM, has to be taken with caution. Eaves and Eaves have shown that CFU-E numbers reach their peak in semisolid cultures of human BM cells between days 7 and 10 [24]. Since in our study we scored all hematopoietic CFC, including CFU-E, between days 14 and 17 of culture, it seems very likely that the CFU-E levels observed by us were suboptimal. This, in fact, may be the reason for the lower proportion of CFU-E, as compared to BFU-E, observed both in UCB and BM samples.

It has been widely recognized that successful hematopoiesis in standard BM D-LTC depends upon the presence of an adherent stromal cell layer, developed simultaneously from the same marrow sample [17]. This adherent cell layer consists of fibroblasts, macrophages, adipocytes and endothelial cells which are capable of producing and secreting both extracellular matrix molecules and cytokines [18, 20, 25-27]. In our study, we observed a poor development of the adherent cell layer in UCB D-LTC. Adherent cell numbers in these cultures were 25%-50% of the numbers observed in BM D-LTC and confluence was never seen. Preliminary morphological analysis of the cell composition of adherent layers indicates that whereas in BM cultures, fibroblasts, macrophages and adipocytes were present, in UCB D-LTC the adherent cell layer consisted almost exclusively of macrophages; fibroblast-like cells were rare and no adipocytes were observed (Gutiérrez-Rodríguez and Mayani, unpublished observations). Indeed, our observations suggest that UCB is a rich source for macrophage development in vitro, since significant numbers of these cells were generated in these cultures. Thus, it is possible that the postulated deficient hematopoietic supportive capacity of the adherent layers of UCB D-LTC is due not only to their reduced cell number, but also to their abnormal cell composition (i.e., absence of fibroblasts). Studies are currently in progress in order to characterize in more detail the cell composition and physiology of the adherent layers developed in UCB D-LTC. It is interesting that Ye and colleagues have recently reported on the establishment of adherent stromal cell layers from human UCB capable of supporting HPC growth for several weeks [28]. The authors cultured UCB mononuclear cells on customized glass coverslips, which show a very different surface (no detectable granular particles or coarse bump structures) from that of standard glass coverslips or standard plastic surface; on the other hand, they maintained their cultures at 37°C for the whole culture period. These conditions clearly differ from the ones used in our study, thus it remains to be determined if the differences in HPC and adherent cell growth observed are due to the differences in culture conditions.

The poor development of the adherent cell layer in UCB D-LTC correlated with the continuous decline in the numbers of HPCs throughout the nine-week culture period, in contrast to BM D-LTC, in which a plateau was observed in HPC numbers between weeks 6 and 9. By week 9, the total number of HPCs in BM D-LTC was 11-fold higher than in UCB cultures. Previous studies have demonstrated that UCB HPCs possess a higher proliferation and expansion potential than BM HPCs when cultured on a preformed marrow stroma [3] or in cultures supplemented with recombinant cytokines [13, 15]. Thus, our observations suggest that the impaired hematopoietic cell growth in UCB D-LTC is due to quantitative and qualitative deficiencies of the adherent cell layer, which is unable to provide the factors required by HPC for their proliferation. This notion has also been supported by the work of Hows and colleagues [3].

It is noteworthy that throughout the culture period a shift towards myelopoiesis was observed, both in UCB and BM D-LTC. Indeed, erythroid and multipotent progenitors disappeared relatively fast in this culture system, so that by weeks 7-9, 100% of the progenitors detected in BM cultures corresponded to myeloid progenitors, whereas in UCB cultures these cells comprised 88%-100% of the total number of progenitors observed. These findings confirm previous studies, showing that in human BM D-LTC myelopoiesis is sustained significantly longer than erythropoiesis [19, 22]. The reason for this is still unclear; attempts to sustain erythropoiesis for longer periods of time by means of adding exogenous erythropoietin have shown only a transient stimulation [22], suggesting that other factors and/or mechanisms necessary for erythroid development are absent in this culture system. In this regard, it is interesting that no significant differences in total erythroid progenitor cell numbers were seen between UCB and BM cultures, even though the former contained significantly lower numbers of adherent stromal cells than the latter. Further studies need to be performed to define the factors and culture conditions necessary for erythroid growth in D-LTC. Similarly, studies aimed at the identification of the factors and conditions necessary for the growth of multipotent progenitors in D-LTC need to be carried out.

It has been shown that most, if not all, of the CFC with high proliferative potential are restricted to the myeloid lineage [29]. It has also been demonstrated that the vast majority of the long-term culture initiating cells exclusively gives rise to progenitors of the granulocyte and/or macrophage lineages [30]. Furthermore, Mayani and Lansdorp have recently demonstrated the presence, at least in UCB, of very primitive HPCs committed to the granulocyte/macrophage lineage (CFU-GM II), whose proliferation and expansion potentials are higher than those of multipotent progenitors [31]. Thus, it is likely that the myeloid progenitors observed both in UCB and BM D-LTC after five weeks of culture are derived from primitive progenitors such as the ones described above. Moreover, it has been shown that these primitive progenitors (i.e., high proliferative potential CFC, long-term culture initiating cells, CFU-GM II) preferentially give rise to mature progenitors of the monocytic lineage; this would explain the shift towards the predominance of CFU-M, both in UCB and BM D-LTC, observed in our study after five weeks of culture. Such a predominance of CFU-M, in turn, correlated with, and was most likely the cause of, the increase in the proportion of monocytes/macrophages both in UCB and BM D-LTC.

In summary, we have thoroughly followed the kinetics of HPC and morphologically recognizable cells in D-LTC established from both UCB and BM. Our study indicates that myeloid progenitor cell growth is deficient in UCB D-LTC, as compared to BM D-LTC, and suggests that this is due to the impaired development of an adherent cell layer, unable to provide the factors and conditions required for their growth. Interestingly, throughout the culture period the total numbers of multipotent and erythroid progenitors were similar both in UCB and BM cultures, regardless of the numbers and quality of the adherent cells present; this suggests that the stroma developed in D-LTC is not sufficient for the proliferation of these progenitors. These results may be relevant not only in our understanding of the biology of UCB-derived hematopoietic and stromal cells, but also in the development of strategies for the ex vivo expansion of UCB HPC.


   Acknowledgments
 
The authors would like to thank Dr. Luis Benitez-Bribiesca, head of the Oncological Research Unit, Oncology Hospital, National Medical Center, for his continuous support. We also thank Dr. Octavio Vera-Morales and the staff from the Gynecology Department of the "1° de Octubre" Hospital, as well as Dr. Fernando Escobedo and the staff from the Gynecology Department of the "20 de Noviembre" Hospital, for making UCB samples available for this study. Dr. Enrique Gómez-Morales and the staff of the Bone Marrow Transplant Unit of the "Bernardo Sepulveda" Hospital, as well as Drs. Armando Mansilla and Guillermo Careaga (Cardiology Hospital, National Medical Center) are also thanked for making normal BM samples available. Some of the culture media used in this study were a generous gift from StemCell Technologies, Inc.; Vancouver, BC, Canada.

This study was supported by grants no. 0122P-M9506 and 0102P-M9506 from "Consejo Nacional de Ciencia y Tecnología" (CONACYT, México) and by grant no. FP0038/79 from "Instituto Mexicano del Seguro Social" (IMSS, México).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication December 4, 1997.



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