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Stem Cells, Vol. 15, No. 4, 305-313, July 1997
© 1997 AlphaMed Press

Importance of Parenchymal:Stromal Cell Ratio for the Ex Vivo Reconstitution of Human Hematopoiesis

Manfred R. Kollera, Ilana Manchel, Bernhard Ø. Palssonb

Aastrom Biosciences, Inc., Ann Arbor, Michigan, USA;
a Present address: Advanced Tissue Sciences, Inc., La Jolla, California, USA;
b Department of Bioengineering, University of California-San Diego, La Jolla, California, USA

Key Words. Cell therapy • Tissue engineering • Bone marrow • Hematopoiesis • Stem cells • Stroma

Dr. Manfred R. Koller, c/o Aastrom Biosciences, Inc., P.O. Box 376, Ann Arbor, MI 48106, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transplantation of living cells and tissues has become an important therapeutic modality for a number of medical conditions. However, insufficient tissue availability and the prohibitive cost of transplantation therapies have hampered their widespread use. New developments in tissue engineering offer the promise of overcoming these limitations, increasing the scope and availability of transplantation therapies [1-4]. Many of these new developments rely on the culture of primary tissues, reconstituting tissue function ex vivo.

Tissues are comprised of functional subunits (e.g., kidney nephrons and small intestinal villi) containing many cell types. These cell types can generally be divided into parenchymal and mesenchymal populations, referring to the embryonic origins of the different cells. Parenchymal populations typically contain the proliferative self-renewing stem cells and their functional differentiated progeny within a tissue, whereas mesenchymal cells constitute the connective or support tissue [5]. In tissue culture systems, the nonparenchymal cell populations, which include mesenchymal and endothelial cells, are collectively referred to as stroma [5]. In addition to its role as support and connective tissue, stroma is known to regulate the function of parenchymal cells through a variety of mechanisms including direct cell-cell contact, soluble cytokines and chemokines, and the formation of extracellular matrix (ECM) [5, 6]. These signals lead to the induction of organogenic processes, such as cell proliferation, differentiation, apoptosis, and migration [7].

Successful tissue culture relies on the simultaneous consideration of physicochemical and biological rate processes [1, 8, 9]. Of particular importance in the latter category is the role of stroma in regulating tissue function and organogenesis [5, 7]. When primary cells are aspirated or biopsied from a donor and placed into culture, they are removed from their normal physiological environment, and respond by trying to reconstitute tissue function in their new environment. If the ex vivo environment simulates the in vivo condition sufficiently well, allowing both the parenchymal and stromal tissue elements to thrive, then partial or full tissue function results [1]. Consequently, the initial conditions of culture with respect to parenchymal:stromal (P:S) cell ratio will play an important role in determining how the culture evolves.

Hematopoietic cell therapies have a long history that includes red blood cell and platelet transfusions, and more recently, bone marrow (BM) transplantation. The use of cultured hematopoietic cells for treatment of patients undergoing cancer chemotherapy has become the subject of much research [1,10-13]. Adult BM, the in vivo site of hematopoiesis, is a very prolific tissue, and hematopoietic cell culture systems have progressed greatly in recent years [1, 14-18]. Enrichment of the primitive hematopoietic parenchymal elements, containing the self-renewing stem cell population, can be performed using cell selection technologies based upon expression of the CD34 surface marker [19]. However, it is known that stromal elements play an important role in determining the fate of developing hematopoietic cells [20, 21], and it has been proposed that specific stem cell supportive niches exist within BM stroma [22]. These known clinical and scientific aspects of hematopoiesis make it a good model system for investigation of the relative roles of parenchyma and stroma in ex vivo human tissue reconstitution.

Previous studies have shown that the inoculum density of mononuclear cells (MNC) [23, 24] and the presence of irradiated preformed stroma (IPFS) have significant effects on cell, colony forming units-granulocyte/macrophage (CFU-GM), and long-term culture- initiating cell (LTC-IC) output from human BM cultures [24], suggesting that P:S ratio is an important culture variable. In order to further explore the relative effects of parenchymal and stromal elements in hematopoietic cultures, experiments must be performed by altering the cellular composition of the inoculum away from its natural composition. The present study utilized a model of growth factor-supplemented human BM culture to study the interaction of parenchymal and stromal cellular elements by systematically varying the cellular composition of the inoculum. CD34-enrichment was used to vary the parenchymal cell content, whereas stromal elements were generated by using either IPFS or CD34-depleted cells from BM. The results demonstrate the strong interactions that occur between parenchyma and stroma, and how such information is useful in the development of optimal hematopoietic cell culture systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture Medium
Long-term bone marrow culture (LTBMC) medium was prepared by supplementing Iscove's modified Dulbecco's medium (IMDM) with 10% horse serum, 10% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all from GIBCO; Grand Island, NY), and 5 µM hydrocortisone (Sigma; St. Louis, MO). Growth medium was prepared by supplementing LTBMC medium with a previously optimized mixture [20] of 2 ng/ml interleukin-3 ([IL-3] Immunex; Seattle, WA), 5 ng/ml GM-CSF (Immunex), 0.1 U/ml erythropoietin ([EPO] Amgen; Thousand Oaks, CA), and 10 ng/ml c-kit ligand ([KL] Immunex).

In some experiments, the growth medium was further supplemented with conditioned medium (CM) taken from cultures of IPFS cells. CM was removed on day 7 and concentrated 10-fold in a Centriprep-10TM device (10 kD cut-off membrane; Amicon; Beverly, MA). This CM concentrate was then used to supplement the growth medium at ten percent (v/v).

Cell Source and Separation Procedure
Human BM cells were obtained with informed consent from iliac crest aspirates, or from processing screens (Baxter Fenwal; Deerfield, IL) obtained after the harvest of BM from normal donors. MNC were collected by Ficoll (1.077 g/ml; Pharmacia; Uppsala, Sweden) separation, and CD34-enriched cells were collected with a MACS laboratory separation system (Miltenyi Biotec; Auburn, CA) as previously described [20].

Flow Cytometry Analysis
Cells to be analyzed were washed and resuspended in phosphate buffered saline ([PBS]; GIBCO) containing 1% bovine serum albumin ([BSA] Intergen; Purchase, NY). Tubes containing 106 cells in 0.5 ml were stained on ice with either PE-HPCA-2 (anti-CD34) or PE-IgG (control) monoclonal antibodies (Becton-Dickinson; San Jose, CA) along with a cocktail of lineage (lin)-specific antibodies: FITC-Leu4 (anti-CD3), FITC-Leu12 (anti-CD20), FITC-LeuM3 (anti-CD15; all from Becton-Dickinson), FITC-anti-CD11b (Serotec; Indianapolis, IN), and FITC-anti-glycophorin A (Dako; Carpinteria, CA). After 15 minutes, cells were washed and resuspended in 0.5 ml PBS/BSA for analysis of CD34+lin– cell percentage on a FACS VantageTM flow cytometer (Becton-Dickinson).

Cell Culture System
Cells were cultured at various densities in 24-well plates (Costar; Cambridge, MA) in 0.6 ml of growth medium, both with and without the presence of various numbers of IPFS cells. IPFS was prepared by trypsinizing adherent stromal cells from primary human BM cultures and irradiating them as previously described [20]. Inoculum densities were always adjusted by the measured CD34+lin– cell purity, so the actual number of parenchymal CD34+lin– cells present in each culture was controlled. In experiments using both CD34-enriched and CD34-depleted cell fractions, the CD34+lin– cell purity in both fractions was measured and used to control the actual number of CD34+lin– cells present. Control experiments showed that IPFS did not proliferate, usually giving a ~70% confluent layer at 5 x 104 cells per well, and that irradiation eliminated hematopoietic parenchymal cells such as CFU-GM and LTC-IC (not shown). After inoculation, 50% of the culture volume was replaced on days 4, 7, 9, 10, and 11. Cells were harvested after 12 days of culture using trypsinization as previously described [20], and were then resuspended in LTBMC medium for counting and assays.

Methylcellulose Colony Assays
Cells were inoculated in colony assay medium containing 0.9% methylcellulose (Dow; Midland, MI), 30% FBS, 1% BSA, 100 µM 2-mercaptoethanol (Sigma), 2 mM L-glutamine (GIBCO), 5 ng/ml PIXY321 (Immunex), 5 ng/ml G-CSF (Amgen), and 10 U/ml EPO. Cells were plated at 3,000 to 20,000 per ml, depending upon the expected clonogenicity. Cultures were maintained for 14 days and were then scored as previously described [20].

LTC-IC Assays
LTC-IC were determined by limiting dilution assay (LDA) on IPFS using a modification [15] of a previously described technique [25]. Briefly, IPFS was prepared as described [20] in 96-well plates at 104 cells per well in 100 µl LTBMC medium. Test cells were then added to these stromal layers at four concentrations in 100 µl LTBMC medium per well (20 replicates each). The plates were then placed at 33°C in a fully humidified atmosphere of 5% CO2 in air, and the cultures were fed weekly by replacing 100 µl LTBMC medium per well. At week 5, cells were harvested from each well as previously described [15]. The cells from each well were added directly to 0.25 ml of colony assay medium in non-tissue culture treated 24-well plates (Falcon; Lincoln Park, NJ). After 14 days, wells were scored for colonies as described above. For each sample, the number of LTC-IC was determined through an iterative calculation procedure [26] based on the maximum likelihood method [27].


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CD34-Enriched Cell Inoculum Density with Fixed IPFS Number
The first set of experiments was designed to determine the effect of CD34-enriched cell inoculum density. These experiments were performed both with and without IPFS (at a single fixed density of 5 x 104 per well). The output of total cells and CFU-GM from CD34-enriched cell cultures without IPFS increased proportionally with increasing inoculum density, such that the total cell and CFU-GM expansion ratios were relatively constant over the range of inoculum densities tested ( Fig. 1). In the presence of IPFS, culture output was significantly higher, but increased less dramatically as the CD34-enriched cell inoculum density was increased ( Fig. 1). Consequently, the cell and CFU-GM expansion ratios declined as the inoculum density of CD34-enriched cells was increased on IPFS. IPFS therefore provided proportionally better support for increasing culture output at low CD34-enriched cell inoculum densities.



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  		Figure 1. Effect of CD34-enriched cell inoculum density on culture output. CD34-enriched cells were inoculated at various CD34+lin– cell densities, both with and without IPFS. The culture output of (A) cells and (B) CFU-GM is shown, along with the calculated expansion ratio of (C) cells and (D) CFU-GM for the different conditions. Each point is the mean of three replicate cultures (±SE) performed within a representative experiment (of eight).

 
Culture output from CD34-enriched cells was significantly increased by the presence of IPFS, consistent with previous reports [20, 28], and the magnitude of this stromal-dependency of CD34-enriched cells within a given experiment has been shown to be dependent upon characteristics of the CD34-enriched cell donor, but not the IPFS cell donor [28]. The present data further show that the stromal-dependency index ([SDI] defined as the ratio of culture output with IPFS:culture output without IPFS) was dependent upon the CD34-enriched cell inoculum density as well ( Fig. 2). For example, at an inoculum density of 1,500 CD34+lin– cells per well, IPFS increased cell and CFU-GM output by 6.3-fold and 15-fold, respectively. At an inoculum density of 6,000 CD34+lin– cells per well, the effect was less pronounced, with IPFS increasing cell and CFU-GM output by 2.4-fold and 4.3-fold, respectively. Note that the CFU-GM SDI is higher than that for total cells, suggesting that stroma exerts more influence on early hematopoietic events [20].



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  		Figure 2. Effect of CD34-enriched cell inoculum density on stromal-dependency. The SDI (fold-increase in culture output due to the presence of IPFS) is shown as a function of CD34+lin– cell inoculum density for the experiment of Figure 1.

 
Effect of IPFS Inoculum Density
Experiments were next performed to examine the effect of IPFS inoculum density at different CD34+lin– cell inoculum densities. As IPFS cell inoculum density was increased, total cell, CFU-GM, and LTC-IC output reached a plateau above 25,000 IPFS cells per well ( Fig. 3). This plateau was achieved regardless of the number of CD34+lin– cells plated, within the range of 1,500 to 6,000 per well, demonstrating that the amount of IPFS required for maximal stimulation was independent of the number of CD34+lin– cells inoculated. Further, the amount of IPFS required for maximal stimulation was similar using IPFS from six different donors.



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  		Figure 3. Effect of IPFS cell inoculum density on culture output. Different numbers of CD34-enriched cells were cultured with different numbers of IPFS cells. The average normalized culture output of (A) cells, (B) CFU-GM, and (C) LTC-IC is shown. Data within each of six independent experiments were normalized with respect to the output obtained using 3,000 CD34+lin– cells without stroma.

 
These data show that the number of IPFS cells required to support CD34+lin– cells did not increase as the number of CD34+lin– cells was increased, suggesting that IPFS did not provide discrete niches for CD34+lin– cells. Such niches would have become limiting as the CD34+lin– cell inoculum density was increased. Instead, the data show that >=25,000 IPFS cells per well were adequate regardless of the number of CD34+lin– cells present. Therefore, an optimal ratio between CD34+lin– cells and IPFS did not appear to exist. Rather, the data suggest a critical cell density effect of IPFS that in turn suggests that IPFS provides soluble signals that build up to effective concentrations in concert with increased IPFS density.

Effect of Soluble Factors Versus Direct Cell-Cell Contact with IPFS
To test the hypothesis just outlined, CM from IPFS was examined for its ability to support the growth of CD34-enriched cells. CD34+lin– cells at different inoculum densities were grown either alone, in medium supplemented with IPFS CM, or directly in contact with IPFS. IPFS CM significantly increased the output of cells and CFU-GM from the CD34+lin– cells over a wide range of inoculum densities ( Fig. 4). However, CM did not completely substitute for the effect of direct contact of CD34+lin– cells with IPFS, because the IPFS-containing cultures always outperformed the cultures supplemented with IPFS CM. The effects of IPFS thus appear to be mediated through both mechanisms: secretion of soluble factors and direct cell-cell contact.



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  		Figure 4. Effect of IPFS and CM from IPFS cells on culture output. CD34-enriched cells at various densities were cultured with 5 x 104 IPFS cells, or in medium supplemented with CM from the IPFS cells. The culture output of (A) cells and (B) CFU-GM is shown. Each point is the mean of three replicate cultures (±SE) performed within a representative experiment (of three).

 
Altering the P:S Ratio in MNC Cultures: Fixed CD34+lin– Cell Number
The experiments described above utilized CD34-enriched cells and IPFS as representative parenchymal and stromal elements, respectively. However, similar experiments can be performed without irradiation of stromal cells by recombining CD34-enriched and CD34-depleted fractions of a BM MNC population as representative parenchymal and stromal elements, respectively. MNC cultures have previously shown to result in greater output than CD34-enriched cell cultures, even when CD34-enriched cells are supported by IPFS [20], making them perhaps the most attractive cell composition for ex vivo hematopoietic cultures. In fact, non-irradiated stroma arising in MNC cultures differs from IPFS, helping to explain the differences in their functionalities. For example, MNC cultures contain significant numbers of CFU-fibroblast, whereas IPFS cultures do not [29], and growth factor production in MNC versus IPFS cultures differs considerably [20].

In order to alter the P:S ratio in these cultures, MNC were processed with a CD34-enrichment column, and both the parenchymal CD34-enriched cells and the stromal CD34-depleted cells were collected. The percentage of CD34+lin– cells was determined in both fractions, and these two cell populations were then recombined in different ratios. Parallel cultures therefore contained the same number (i.e., 3,000) of CD34+lin– cells, but with different numbers of CD34– stromal cells. Unmanipulated MNC, containing 3,000 CD34+lin– cells, were cultured in parallel as a control. The P:S ratio was shifted both above and below the native ratio present in the unmanipulated MNC mixture.

As the P:S ratio was increased above the native level, culture output decreased rapidly, even though the number of CD34+lin– cells remained constant ( Fig. 5). At the native P:S ratio, the mixture of CD34-enriched and CD34- depleted cells gave slightly lower output than unmanipulated MNC (p < 0.05, paired t-test), suggesting that some important CD34– cellular elements may have been depleted in the separation. When the relative P:S ratio was lowered to 0.5, the performance was again equivalent to unmanipulated MNC (p = 0.4), presumably due to the restoration of the depleted CD34– elements. Further lowering of the relative P:S ratio gave higher cell output, but decreased CFU-GM and LTC-IC (p < 0.05) output. Therefore, the optimal P:S ratio was very near the native BM composition.



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  		Figure 5. Effect of parenchymal:stromal (P:S) cell ratio on MNC culture output, using a fixed number of CD34+lin– cells. MNC were separated into CD34-enriched and CD34-depleted fractions, and were recombined in different ratios to yield different P:S cell ratios relative to the native MNC population (defined as 1.0). The average normalized culture output of (A) cells, (B) CFU-GM, and (C) LTC-IC is shown. Data within each experiment were normalized with respect to the output obtained using CD34-enriched and CD34-depleted fractions at the native MNC ratio. Each point is the normalized mean (±SE) of four independent experiments, each performed with three replicate cultures. The filled circle represents the unmanipulated MNC control culture.

 
Altering the P:S Ratio in MNC Cultures: Fixed Total Cell Number
Another method for altering the P:S ratio is to use a fixed total inoculum cell number with varying ratios of CD34+lin– and CD34– cells. This was accomplished by recombining CD34-enriched and CD34-depleted cells together in different ratios, this time maintaining the total inoculum cell number at 5 x 105 per well. As the percentage (and total number) of CD34+lin– cells was increased within the mixture, the output of cells reached a plateau ( Fig. 6), representing the maximum cell density that can be supported by this culture system [23, 24]. In contrast, CFU-GM output increased in nearly a linear fashion throughout the range tested, suggesting that CFU-GM supportive elements were not limiting as the number of CD34+lin– cells increased and the number of stromal cells decreased. Unlike CFU-GM output, the output of LTC-IC reached a plateau, suggesting that stromal elements became limiting for the support of this primitive cell population.



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  		Figure 6. Effect of parenchymal:stromal (P:S) cell ratio on MNC culture output, using a fixed total cell number. MNC were separated into CD34-enriched and CD34-depleted fractions, and were recombined in different ratios to yield different CD34+lin– cell percentages within the fixed total cell number. The average normalized culture output of (A) cells, (B) CFU-GM, and (C) LTC-IC is shown. Data within each experiment were normalized with respect to the output obtained using CD34-enriched and CD34-depleted fractions at the native MNC purity (mean 2%). Each point is the normalized mean (±SE) of seven independent experiments, each performed with three replicate cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tissue function depends on interactions between multiple cell types, whereas purified cell populations exhibit functionalities of that particular cell type. The normal physiological functions of tissue, tissue formation, and tissue repair are strongly dependent on interactions between parenchyma and stroma. In the present study, a model system of human hematopoietic tissue function was used to study these interactions. The preparation of BM parenchymal and stromal elements was based on established technologies using IPFS, CD34-enriched cells, and CD34-depleted cells. The data showed that: A) IPFS was important and qualitatively changed the effects of CD34+lin– cell inoculum density; B) the effect of IPFS titrated with cell number regardless of the CD34+lin– cell number present, suggesting a soluble growth factor mechanism; C) CM experiments showed that IPFS activity was soluble, but cell-cell contact may be important for inducing the production of these soluble factors (see below); D) the native P:S ratio in MNC gave the best culture performance, and E) LTC-IC supportive niches arising within MNC stroma appear to be limited in number.

The importance of stroma in tissue function, and in hematopoietic cell culture in particular, is well documented [5, 20, 22, 30, 31]. The effect of IPFS on the growth of CD34-enriched cells was clearly shown. The stromal dependency of CD34-enriched cells was high, and interestingly, dependent on the inoculum density of CD34+lin– cells. CD34+lin– cells may communicate in a cell density-dependent fashion that signals the need for increased cell production. The need for increased cell production may physiologically be expected to be dependent on cell density, and a reasonable mechanism for such a signal is via a soluble pathway. The production of soluble Epo by the kidney in response to low red blood cell count is such an example [32].

The activity of IPFS was found to be titratable, and above a critical IPFS density, the supportive effects of IPFS were maximal regardless of the number of CD34+lin– cells present. This result is similar to what is observed in the titration of a soluble growth factor, suggesting that the effects of IPFS were mediated through soluble factors. Direct examination of IPFS CM confirmed this expectation. However, these experiments also showed that soluble signals are not the only means of communication between IPFS and CD34+lin– cells, because direct cell-cell contact effects were discernible. For example, IPFS CM did not increase culture output to the same extent as direct contact. One explanation which is consistent with these observations is that the presence of CD34+lin– cells on IPFS induces production of additional soluble activity from IPFS [33]. This induced activity, dependent on CD34+lin– cell-IPFS cell direct contact, in turn acts on all CD34+lin– cells present in the culture through a soluble mechanism, thereby overcoming potential limitations in IPFS cell contact sites. Other coculture systems have been shown to exhibit two-way, contact-mediated communication [34, 35], and the induction of soluble IL-6 from osteoblasts by CD34+ cells has also been recently reported [36], further supporting this concept.

It has been proposed that stroma provides discrete niches within which immature hematopoietic cells are maintained [22], consistent with the observation that effective BM transplantation requires ablation of host hematopoiesis to achieve donor engraftment. However, other experimental systems suggest that ablation may not be necessary to achieve engraftment [37, 38], arguing against limiting niches. The present data indicate that there was not a key ratio between IPFS and CD34+lin– cells, suggesting that there were no limiting niches in the IPFS environment. Much of the effect of IPFS appears to be mediated through soluble factors, although CD34+lin– cell contact is required to induce the full soluble activity from IPFS [33]. In contrast, direct contact with MNC stroma is significantly more potent than MNC CM [33]. The data presented here with MNC stroma suggest that LTC-IC supportive niches were limiting as the CD34+lin– cell number was increased. These observations, along with the fact that MNC stroma supports LTC-IC to a greater extent than IPFS [20], suggest that a limited number of LTC-IC supportive niches are present in MNC stroma, whereas IPFS lacks these niches and acts on LTC-IC predominantly through a less potent soluble mechanism.

In contrast with the results for LTC-IC output, limiting niches for CFU-GM support were not evident. Because progenitors and mature hematopoietic cells are numerous and cycle at a fast rate in BM tissue [39, 40], rare stromal elements would likely be too limiting to support the process entirely through a direct contact mechanism. Instead, stroma provides soluble signals, some of which are known to prevent apoptosis and permit maturing cells to complete their development [41, 42]. For stem cells, which are few in number and cycle at a very slow rate [40], the possibility of direct cell contact as the dominant regulatory mechanism is more likely. Primitive cells within certain stromal niches may be prevented from going into cycle and/or differentiating to the progenitor cell stage [31, 43, 44], thereby preventing excessive mitosis and/or apoptosis. Too high a rate of commitment of stem cells to differentiation has at least two important consequences in a tissue culture system. The first is a loss of stem cell potential by a wasteful differentiation strategy: too many cells would be made, only to die. The second consequence is a loss of stem cells, which is more serious in terms of clinical utility. The complete absence of stroma and stroma-derived signals appears to lead to the loss of human stem cells in culture [20], and murine studies have confirmed this observation, demonstrating that cultures of purified stem cells quickly lose in vivo repopulating potential [21].

The present results have general implications for tissue reconstruction ex vivo. The presence of stromal elements is important for tissue function, and parenchymal cell behavior is greatly influenced by the quantity and quality of stromal cells present. The requirement for accommodation of functional stroma complicates the design of clinical-scale tissue culture systems. Another consideration for cell culture system design and operation involves the control of soluble signals. Positively acting soluble factors produced by stroma must be allowed to accumulate to a sufficient degree, although waste products and negatively acting factors must be removed. A proper balance can be achieved by finding the optimal volume and medium exchange rate for the culture system, and at present, satisfying these requirements is an empirical process that is perhaps best accomplished using a multidimensional, statistically designed experimental approach [24].


   Acknowledgments
 
We thank Mahshid A. Palsson and Robert J. Maher for excellent technical assistance and David A. Brott for flow cytometry analysis. We also thank Drs. Alan Smith and Beverly Lundell for critical reading of the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication April 20, 1997.




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