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Stem Cell Res. Author manuscript; available in PMC Jan 1, 2010.
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Enumeration of the colony-forming units–fibroblast from mouse and human bone marrow in normal and pathological conditions

Abstract

Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units–fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 ± 1.0 to 11.5 ± 4.0 per 1 × 105 nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 ± 4.1 for children and 32.3 ± 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology.

Introduction

Bone marrow stromal cells [BMSCs, renamed in the1990s as “mesenchymal stem cells” (Caplan, 1991)] contain a sub-population of multipotential skeletal stem cells (SSCs) that can differentiate toward multiple skeletal tissues including bone, cartilage, fat, and hematopoiesis-supportive reticular stroma (Bianco and Robey, 2004; Friedenstein, 1980). Due to the BMSC capacity for ample ex vivo expansion, and the presence of SSCs within their populations, BMSCs have become a centerpiece of innovative therapeutic approaches such as tissue engineering and gene therapy (Bianco et al., 2001; Krebsbach et al., 1999). Far less recognized is BMSC/SSC involvement in skeletal and marrow-related pathologies. Yet when BMSCs/SSCs carrying natural or targeted genetic changes are transplanted in vivo into the subcutis of immunodeficient mice, they generate ectopic ossicles that recapitulate pathological features of the original lesion; thus, abnormal BMSCs/SSCs can single-handedly re-create a disease (Bianco et al., 2000; Holmbeck et al., 1999; Kuznetsov et al., 2004; Riminucci et al., 2001). Moreover, in several bone disorders, such as fibrous dysplasia of bone, hyperparathyroidism, and early stages of Paget disease, distinct changes in the number, organization, and apparent function of marrow stromal cells are as prominent as the changes in bony structures themselves (Bianco and Robey, 1999; Robey and Bianco, 1999). Consequently, analysis of marrow stroma, both qualitative and quantitative, provides another way to probe pathogenetic mechanisms related to SSC activity in both humans and transgenic animals.

In low-density bone marrow cell cultures, discrete colonies of adherent fibroblast-like BMSCs are formed, each colony arising from a single precursor cell termed a colony-forming unit–fibroblast (CFU-F) (Friedenstein, 1973, 1976; Friedenstein et al., 1978; Latsinik et al., 1986). The number of colonies formed per a definitive number of marrow cells plated, the colony-forming efficiency (CFE), thereby defines the CFU-F content among marrow cells, representing in vitro enumeration of a clonogenic subset of in vivo marrow stromal cell populations. Furthermore, it has been demonstrated that CFU-F populations are not homogeneous but rather contain a hierarchy of progenitors including multipotential SSCs and committed progenitors. By in vivo analyses of clonal strains established by single CFU-Fs, SSCs constitute from one-tenth to one-third of all CFU-Fs (Bennett et al., 1991; Chailakhian et al., 1978; Gerasimov Iu et al., 1986; Kuznetsov et al., 1997b, 2004). To date, there are no markers that can be used prospectively to separate multipotential CFU-Fs, or SSCs, from more committed CFU-Fs. Thus, the CFE assay is currently the closest approximation to the determination of the concentration of SSCs in bone marrow (Bianco and Robey, 2004).

CFE values are based on in vitro proliferation of CFU-Fs and their descendents and are, therefore, substantially dependent on the culture conditions. To optimize the parameters of the CFE assay, we first analyzed the effects of a major component of cell culture media, fetal bovine serum (FBS), on BMSC colony formation. We found for the first time that in many different lots, heat inactivation had a deleterious effect on CFE in mouse and human cultures. We then extended our determination of CFE using several inbred mouse strains and transgenic lines with substantial bone involvement, as well as normal human donors and patients with several skeletal, metabolic, and hematological disorders, in which CFE has not been previously described. This study was, by design, limited to the analysis of BMSC colony formation and CFU-F concentration under a variety of conditions. Our results indicate that, under appropriate culture conditions, CFE values are considerably and variably affected in various disease states in mice and humans.

Results

Comparison of the effects of different lots of FBS on murine and human CFE

We studied the effects of 11 FBS lots (non-heat-inactivated) on BMSC colony formation using a mouse bone marrow cell suspension prepared from two FVB/N mice (6-week-old females) and a human bone marrow cell suspension from a single normal donor (normal adult donor 8, 76-year-old female, Table 1). Depending on the FBS lot, mouse CFE varied from 4.3 to 7.5 per 1 × 105 nucleated marrow cells (Fig. 1A). The two “worst” lots (Nos. 1 and 2) supported significantly lower CFE than the three “best” lots (Nos. 5, 7, and 9), while lots 3 through 11 generated CFE values not significantly different from each other. Human CFE varied from 12.9 to 40.6 per 1 × 105 nucleated marrow cells (Fig. 1B). Similar to mouse cultures, the two “worst” lots (3 and 5) supported significantly lower CFE than all remaining lots, which generated CFE comparable to one another. Interestingly, lot 5 was among the most effective in mouse cultures but was among the least effective in human cultures. Vice versa, lot 2 supported the highest human CFE but the lowest mouse CFE.

Figure 1
Mouse and human CFE in medium with various FBS lots. Mouse and human bone marrow cells were plated in medium with FBS of 1 of 11 lots, either noninactivated or heat inactivated. (A) Mouse cultures. Mouse bone marrow cells from two FVB/N mice were plated ...
Table 1
Normal donors

Effect of FBS heat inactivation

In mouse cultures, heat inactivation reduced CFE in all FBS lots studied; for four lots, the reduction was statistically significant (Fig. 1A). In human cultures, heat inactivation significantly decreased CFE in six FBS lots, including two lots (3 and 4) that totally lost their ability to support BMSC colony formation (Fig. 1B). In no instance did heat inactivation increase CFE in murine or human cultures. The degree of heat inactivation-induced loss of activity did not correlate with the original potency of a lot prior to inactivation (compare lots 3, 4, 5, and 6, Fig. 1B). For some lots, the relative loss of activity differed substantially in mouse and human cultures. For example, heat inactivation of lot 9 did not affect its activity in mouse cultures but caused a twofold CFE decrease in human cultures (Figs. 1A and 1B). We then asked if the effect of heat inactivation would vary, not only between species, but between individual bone marrow donors as well. Heat inactivation of a single FBS lot (No. 1) significantly reduced CFE in cultures of one human donor but not in those of four others (Fig. 2).

Figure 2
Human CFE in cultures from several donors with noninactivated and heat-inactivated FBS. Bone marrow cells from five donors were plated in triplicate in medium containing FBS lot 1, either noninactivated or heat inactivated. The differences between the ...

CFE in different murine strains

Using optimized non-heat-inactivated FBS, CFE varied considerably between inbred mouse strains, with the highest CFE (mean ± SEM) of 11.5 ± 4.0 per 1 × 105 nucleated marrow cells in Rosa mice and the lowest CFE of 3.5 ± 1.0 in CBA mice (Fig. 3A). In transgenic mice featuring profound phenotypes, CFE was radically changed compared with that of their respective wild-type littermates: increased in MT1-MMP-deficient mice and in mice with an FGFR3 mutation and reduced in Col1-caPPR mice (as we reported previously in Kuznetsov et al., 2004) and in IL-5-overproducing mice (Fig. 3B).

Figure 3
CFE in mouse cultures. (A) CFE in cultures from various strains of inbred mice. Mouse bone marrow cells were plated in triplicate. Numbers of animals analyzed per strain were CBA, 11; Rosa, 4; FVB/N, 12; C57Bl, 5; NIH-bg-nu/nu-xid, 2. Only differences ...

Normal human CFE

In cultures of normal bone marrow from 16 children, ages 6 months to 15 years, CFE varied from 11.3 to 101.3 per 1 × 105 nucleated cells (Table 1), averaging 52.2 ± 4.1 (mean ± SEM). In specimens of normal bone marrow from eight adults, ages 29 to 76 years, CFE ranged from 12.3 to 56.8 per 1 × 105 nucleated cells (Table 1), averaging 32.3 ± 3.0. Linear regression analysis revealed a statistically significant inverse correlation between CFE and the age of normal bone marrow donors (regression value, r = −0.41, P < 0.05, Fig. 4). However, the age-related CFE decline was relatively modest and did not represent changes in orders of magnitude. For male (n = 7) and female (n = 9) pediatric donors, CFE (mean ± SEM) was, respectively, 60.3 ± 11.5 and 44.7 ± 9.7, P > 0.05. For male (n = 2) and female (n = 6) adult donors, CFE was 41.3 ± 15.6 and 28.7 ± 4.6, respectively, P > 0.05.

Figure 4
Correlation between CFE and age in normal human donors.

Pathological human CFE

Among pediatric patients, CFE was drastically decreased in a patient with achondroplasia (severe achondroplasia with developmental delay and acanthosis nigricans, or SADDAN) and elevated in a patient with sickle cell anemia and in one of two patients with Proteus syndrome. CFE was normal (P > 0.05 in comparison with a group of normal pediatric donors) in patients with Marfan syndrome, Sprengel deformity, or Blount disease and in a second patient with Proteus syndrome (Table 2, Fig. 5A). In adult patients, CFE was radically reduced in two patients with congenital generalized lipodystrophy and in a patient with pseudoachondroplasia, significantly decreased in a patient with Paget disease and in two of three patients with Job syndrome, and increased in a patient with alcaptonuria. It was normal in both patients with familial partial lipodystrophy and in a third patient with Job syndrome (Table 2, Fig. 5B).

Figure 5
CFE in cultures from normal and pathological human donors. (A) Pediatric patients <18 years of age. (B) Adult patients >18 years of age. Each black bar represents a single donor, an average of three or four cultures. All differences are ...
Table 2
Pathological donors

Discussion

The identity of CFU-Fs

Marrow stromal precursor cells, also termed CFU-Fs or cells giving rise to BMSC colonies in vitro, are thought to arise from cells with vague morphology and multiple names: adventitial cells, reticular cells, stromal fibroblasts, stromal cells, bone marrow stromal cells (in situ), preadipocytes, and Westen–Bainton cells. These cells form a subendothelial (adventitial) layer of sinusoidal walls and project away extensive processes that are associated with hematopoietic cells (Bianco and Riminucci, 1998). They express low levels of collagen types I and III and osteonectin and are, therefore, considered fibroblastic (Bianco et al., 1999; Owen and Friedenstein, 1988). These cells also coexpress alkaline phosphatase (Westen and Bainton, 1979), endoglin (CD105), and high levels of CD146, the latter being a major hallmark of all CFU-Fs in vitro and of subendothelial cells in vivo (Sacchetti et al., 2007). After marrow cell suspension is plated in vitro, those reticular cells that attach to the culture vessel and begin to proliferate to form BMSC colonies are, in fact, CFU-Fs (Friedenstein, 1990; Friedenstein et al., 1990). Thus, the CFE reflects the prevalence of stromal clonogenic precursors, or CFU-Fs, in bone marrow. Important to this point, accurate determination of CFE is highly dependent on the ability of the culture conditions to support proliferation of CFU-Fs and their descendants; suboptimal conditions may permit only a proportion of existing CFU-Fs to form colonies. Consequently, we analyzed the influence of culture conditions on BMSC colony formation and then used optimized conditions to study the variability of CFU-F concentration in various mouse and human marrow cell populations.

The use of terms and abbreviations: CFU-F, BMSC, SSC, MSC

At this point, it seems appropriate to discuss the terminology used for fibroblast-like cells in adherent bone marrow cell cultures and demonstrate that the terms used in this paper are neither redundant nor interchangeable. Indeed, “colony-forming unit–fibroblastic,” or CFU-F, is an in vitro term encompassing those members of an in vivo Westen–Bainton cell population that, under actual culture conditions, form adherent colonies. The original CFU-Fs, as such, cease to exist after the first cell division in vitro. The colonies formed by CFU-Fs, as well as the ensuing passaged cell lines, consist of bone marrow stromal cells (in vitro), or BMSCs; this term, thus, represents culture-derived progenitors of CFU-Fs. By clonal analysis, it was demonstrated that only some CFU-Fs have stem cell properties, capable of both extensive proliferation and in vivo differentiation into multiple skeletal cell types: osteoblasts, chondrocytes, adipocytes, fibroblasts, and adventitial reticular cells. Such CFU-Fs are thus termed “skeletal stem cells,” or SSCs (Bianco et al., 2008); the continuous existence of SSCs among BMSC populations renders the latter multipotential. It is not known what part of BMSCs are SSCs: certainly, less than 20–30% that are normally clonogenic, or able to form secondary colonies, since clonogenicity is necessary but not sufficient to grant stem cell status. This question may be directly answered by clonal analysis of secondary BMSC colonies.

Contrary to the three above-mentioned terms, the widely used term “mesenchymal stem cells,” or MSCs, seems inappropriate for several reasons. First, MSC populations are heterogeneous, with only some of these cells possessing stem cell properties; it is, therefore, misleading to call all of them “stem cells.” Second, the nonskeletal mesenchymal potential of MSCs (ability to create tissues such as skeletal muscle, myocardium, and smooth muscle) has not been formally proven in vivo (Bianco et al., 2008). Thus, the majority of the “mesenchymal stem cells” are, in fact, neither stem nor mesenchymal. Finally, the term “MSCs” is inconvenient because, contrary to “BMSCs,” it carries no information regarding the tissue origin of the cells.

Variability of FBS lots

It has long been practical knowledge that growth-promoting activity varies between FBS lots, but analysis of the molecules underlying the differences in such a complex mixture as FBS is not yet feasible. This causes laboratories to perform routine FBS trials and to use “preselected” lots thereafter (Lennon et al., 1996; Mannello and Tonti, 2007). Here, we document that FBS lots do, indeed, vary broadly in their support of BMSC colony formation and that the relative quality of the lots is a species-related phenomenon. Certain lots inferior in mouse cultures were among the best in human cultures and vice versa. Our data indicate that FBS testing needs to be performed separately for BMSCs from each species under study.

FBS heat inactivation: why is it harmful for BMSC colony formation?

To our knowledge, this report is the first formal comparison between heat-inactivated and non-heat-inactivated forms of FBS as related to BMSC colony formation. Heating serum, as a means of complement inactivation and prevention of complement-mediated cell lysis, has been regarded as a mandatory step for a wide range of cell cultures (Giard, 1987; Leshem et al., 1999). Yet FBS heat inactivation was found to be unnecessary for immune responses in vitro, such as lymphocyte proliferation, IL-2 production, and cell-mediated cytotoxicity (Leshem et al., 1999), as well as for cell attachment to plastic (Giard, 1987). Beyond such limited data, the effect of FBS heat inactivation on cell behavior has long been neglected (Mannello and Tonti, 2007). Our previous studies have always utilized non-heat-inactivated FBS based on early work by Friedenstein and coworkers (Friedenstein, 1990; Friedenstein et al., 1990). In this study, using both mouse and human BMSC colony formation assays, not a single FBS lot improved its performance following heat inactivation. On the contrary, many heated lots demonstrated significantly reduced ability to promote BMSC colony formation. The degree of CFE reduction was not dependent on the original potency of a lot and was both species- and donor-related. Our findings imply that for both mouse and human BMSC colony formation, heat inactivation of FBS is contraindicated. Keeping in mind that the formation of BMSC colonies by CFU-Fs represents the first, crucial step of BMSC in vitro generation, the same conclusion will most likely apply to the entire process of mouse and human BMSC ex vivo expansion. Our results may be explained by the fact that heat inactivation reduces the concentrations of several serum growth factors, including PDGF-BB and HGF (Ayache et al., 2006), although neither any known growth factor alone nor any combination of growth factors can substitute for FBS (Kuznetsov et al., 1997a). In addition, the presence of intact complement in noninactivated FBS may cause little or no harm because, on one hand, FBS contains just a fraction of adult levels of all complement components and, on the other hand, heating has a dual effect of decreasing titers of C1, C2, C7, and C8 but increasing titers of C3 and C6 by inactivation of a heat-labile inhibitor (Linscott and Triglia, 1981).

Our data indicate that BMSC populations generated in medium with heat-inactivated and noninactivated FBS may differ with respect to their proliferation histories. It would be interesting to analyze whether these two populations vary in other fundamental aspects, such as differentiation potential, proliferation ability, or cell surface characteristics. Such comparison, however, would represent a much broader approach than that employed in this study, which was, by design, limited to the analysis of BMSC colony formation and CFU-F concentration under a variety of conditions.

Mouse CFE: variability and its possible causes

Using optimized culture conditions, we found CFE to range from 3.5 to 11.5 per 1 × 105 nucleated marrow cells in inbred mouse strains. It needs to be acknowledged that the precise physiological basis of such CFE variability is currently not clear; in other words, the observed variations in CFU-F numbers cannot be correlated with any apparent biological difference related to bone or marrow biology in different mouse strains. It has been shown, however, that CFU-F numbers in rats correlate with changes in bone anabolic activity observed after ovariectomy and the administration of bone anabolic drugs (Nishida et al., 1994; Scutt et al., 1996). It is also well recognized that mouse strains show considerable variation in the quantity and biomechanical quality of their bones independent of aging (see Jepsen et al., 2003, for example). While biomechanical bone quality is often related to architectural features that vary between mouse strains, it is also possible that the quantity and quality of bone can result from changes in the number and activity of stromal cells/SSCs. Earlier, CFE was shown to vary 10-fold depending on the mouse strain, from 0.03 to 0.3 per 1 × 105 nucleated marrow cells (Phinney et al., 1999). While the finding of strain-related variability is consistent with our data, the extremely low CFE values observed in the aforesaid study imply the apparent loss of 90 to over 99% of CFU-Fs. This loss can be explained by substandard culture conditions employed, such as very high plating density (2.5 × 107 cells per 9.5 cm2, or 65 times higher than in the current paper) and lack of irradiated marrow cells.

In addition to inbred mouse strains, our study also included four lines of transgenic mice. In all of them, CFE was considerably altered, either increased or decreased, in comparison with their respective wild-type littermates; such dramatic CFE changes corresponded well to profound bone phenotypes. Interestingly, in mice bearing an activating FGFR3 mutation (G369C) (Chen et al., 1999), CFE was altered in a direction opposite that of a patient with SADDAN caused by another activating FGFR3 mutation (K650M) (Iwata et al., 2001).

Normal human CFE and the need for adequate culture conditions

In normal human marrow, the average CFE was 52.2 for children and 32.3 for adults. Our values for normal human CFE are in agreement with some earlier publications (Akintoye et al., 2006; Doucet et al., 2005; Kuznetsov and Gehron Robey, 1996; Kuznetsov et al., 2000; McIntyre and Bjornson, 1986; Nagao et al., 1983b; Vladimirskaia et al., 1989), but are considerably higher than CFE values reported elsewhere: below 1 (Beresford et al., 1994), around 1 (Vladimirskaia, 1977), or below 3 per 1 × 105 nucleated marrow cells (Bernardo et al., 2007; Oreffo et al., 1998a, 1998b). Such diminutive CFE values suggest, once again, that over 90% of the existing CFU-Fs were unable to form colonies because of inadequate conditions of either cell preparation (density gradient centrifugation during which CFU-Fs may be lost) or cell culture (most probably a substandard serum). It must be emphasized that it is not known whether any particular subset of CFU-Fs is preferentially lost in cultures with low CFE. However, SSCs, which are deeply quiescent in vivo, may be critically dependent upon strong growth factor stimulation to initiate colony formation; they may therefore be the first subset to be lost under substandard conditions. In this study, an inverse correlation between normal human CFE and donor age was observed. This is in agreement with the majority of earlier publications (D'Ippolito et al., 1999; Galotto et al., 1999; Kolesnikova et al., 1978; Mets and Verdonk, 1981; Nishida et al., 1999; Rogova et al., 1981), but contradicts several others (Oreffo et al., 1998a, 1998b; Stenderup et al., 2001). The slight decrease that we noted with aging may be related to a slower rate of fracture healing in the elderly.

Question of reproducibility of human CFU-F enumeration

In this study, only a single marrow specimen could be obtained from the majority of donors, not allowing us to measure directly the reproducibility of human CFU-F enumeration. In two cases, however, in which repeated biopsies from the same donor were available, 10 and 12 months apart, close CFE values were received (not shown). A possible source of CFE variability could also be due to different CFU-F concentrations in various parts of the skeleton. In this study, most adult specimens, both normal and pathological, were obtained from the iliac crest, thus eliminating the concern of location-caused variability. Specimens from most normal pediatric donors were also from the iliac crest; however, marrow from each pathological pediatric donor was obtained from a different site of the skeleton (Table 2). According to earlier publications, CFE values in human donors may, indeed, vary depending on the part of the skeleton; values for iliac crest differ from those for sternum, femur, tibia, maxilla, and mandible by the factor of 1.5 to 3.5 (Akintoye et al., 2006; Astakhova and Panchenko, 1991; Suzuki et al., 2001). Meanwhile, in this study, CFE values for pathological pediatric donors differed from normal values by a factor of 47 [achondroplasia (SADDAN)] or 12 (sickle cell anemia), considerably surpassing a possible variability caused by location.

Human CFE in various pathological conditions: a review of the literature

Postnatal stromal cells/SSCs are central mediators of skeletal homeostasis owing to their participation, not only in new bone formation, but also in the control of bone resorption (Bianco and Robey, 2004). It has been our hypothesis that any genetic defect (intrinsic factor) or change in the microenvironment (extrinsic factor) that alters the numbers or the biological activity of stromal cells/SSCs will lead to skeletal abnormalities (Bianco and Robey, 1999). In a review of the literature, we noted that CFE was decreased in patients suffering from alcoholism, a condition associated with reduced bone mass and osteoporosis (Giuliani et al., 1999). In patients with osteoporosis per se, however, CFE was normal (Oreffo et al., 1998a; Stenderup et al., 2001), as it was in patients with osteoarthritis of the hip or knee (Majors et al., 1997; Oreffo et al., 1998a, 1998b). Decreased CFE characterized patients with delayed fracture healing (Zaritskii et al., 1983), nonunions (Seebach et al., 2007), and complicated or unsuccessful recovery after surgery for osteomyelitis (Astakhova, 1988). It has been reported that CFE is higher in males than in females, and that in females, it is increased after multiple fractures (Seebach et al., 2007).

Owing to the central role that stromal cells/SSCs play in the support of hematopoiesis (Bianco and Robey, 2004), changes in CFE may be reflective of not only diseases of bone tissue proper, but also hematopoietic disorders, even in the absence of overt histological changes in bone, or stroma, which is not easily imaged. CFE was shown to be normal in some hematological disorders (Bianchi Scarra et al., 1983; Domracheva et al., 1980, 1981a, 1981b; Hirata et al., 1989; Kaneko et al., 1982) and decreased in others (Carlo-Stella et al., 1997; Domracheva et al., 1981a, 1981b, 1982b, 1984; Duhrsen and Hossfeld, 1996; Hirata et al., 1986; Hotta, 1983; Kaneko et al., 1982; Katsuno et al., 1986; Nagao et al., 1981, 1983a; Nara et al., 1984), while under certain conditions, it varied depending on the stage of a disease (Hirata et al., 1986; Kaneko et al., 1982; Katsuno et al., 1986). CFE values were found to have prognostic significance in patients with acute leukemia (Hirata et al., 1986; Katsuno et al., 1986) and Hodgkin disease (Baisogolov et al., 1973, 1976; Rudakova, 1978). More recently, decreased CFE was observed in patients with untreated lung carcinoma (Chasseing et al., 1997a) and breast cancer (Chasseing et al., 1997b), as well as following chemo/radiotherapy in bone marrow transplant recipients (Galotto et al., 1999), particularly in those suffering from acute graft versus host disease (Okamoto et al., 1991). Notably, low CFE correlated with reduced bone mineral density in transplant recipients (Galotto et al., 1999). The findings related to diseases of both bone and hematopoietic marrow demonstrate the potential importance of CFE for both prognosis and therapeutic choices in these conditions and highlight the need for accurate determinations of CFE.

Human CFE in several pathological conditions: novel findings

Our study describes, for the first time, CFE values for several skeletal, metabolic, and hematological pathologies. CFE was dramatically decreased in patients with congenital generalized lipodystrophy, pseudoachondroplasia, achondroplasia (SADDAN), and Paget disease of bone and radically increased in patients with alcaptonuria and sickle cell anemia. CFE values were mixed (normal in some but abnormal in others) in patients with Job syndrome and Proteus syndrome; it is now recognized, however, that patients diagnosed with Proteus may have various subtypes of the syndrome. CFE was normal in patients with Marfan syndrome, Sprengel deformity, Blount disease, and familial partial lipodystrophy. A decrease in CFE in congenital generalized lipodystrophy may reflect changes in the hematocrit that have been observed in these patients (personal communication, Dr. Philip Gorden, NIDDK, NIH), whereas the increased CFE noted in the sickle cell anemia patient may very well reflect the need for an increased support of hematopoiesis by stromal cells/SSCs, based on the destruction of abnormal blood cells. In late-stage Paget disease, a decrease in CFU-F numbers could be the result of multiple rounds of bone turnover leading to SSC depletion. The potential role of stromal cells/SSCs in the other disorders studied here is not immediately apparent, but warrants further investigation. Despite the lack of in-depth analyses of the human diseases studied, our current data have a broader implication demonstrating that in a substantial number of skeletal, metabolic, and hematological disorders, CFE, indicative of stromal cell/SSC content, is considerably and variably affected, reflecting the disturbed status of the bone marrow stromal compartment. It would be of interest to determine if abnormal CFE values described for these bone- and marrow-related pathologies could serve as diagnostic and prognostic factors, as well.

Conclusions

In conclusion, marrow CFU-F numbers were analyzed in a series of normal and transgenic mice, as well as in normal human donors and in patients with several skeletal, metabolic, and hematological pathologies. Our findings indicate that CFE values, which critically rely on the use of appropriate culture conditions, are substantially abnormal in many pathologies and may provide useful insights into pathogenetic mechanisms of bone/bone marrow disorders.

Materials and Methods

Bone and marrow specimens

Bone marrow from 2- to 6-month-old CBA, FVB/N, Rosa 26 [BALB/cJ-TgR(ROSA26)26Sor], C57Bl/6, and NIH-bg-nu/nu-xid female mice and 5- to 10-week-old Hartley male guinea pigs (Charles River Laboratories, Raleigh, NC, USA) was used in mouse experiments. Additionally, marrow from 2- to 4-month-old transgenic mice featuring profound abnormalities in bone structure and metabolism, and from their wild-type littermates, was studied. The four transgenic lines were MT1-MMP-deficient, mice lacking membrane-bound matrix metalloproteinase (Holmbeck et al., 1999); IL-5-overproducing, mice constitutively overexpressing IL-5 from T cells (Macias et al., 2001); Col1-CaPPR, mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer (Calvi et al., 2001); and mice harboring activating FGFR3 mutation (G369C) (Chen et al., 1999). Animals were sacrificed by CO2 inhalation in compliance with an institutionally approved protocol for the use of animals in research (Protocol 00-113). Femora, tibiae, and humeri were aseptically removed and cleaned of adherent soft tissues.

Specimens of human bone were received in accordance with NIH regulations governing the use of human subjects under Protocols 94-D-0188, 97-D-0055, 98-D-0145, and 01-D-0184. Normal bone was obtained from either healthy volunteers who received a bone biopsy or as surgical waste from individuals undergoing surgery for trauma or corrective surgery (Table 1). Patients who had proven or presumable bone/bone marrow pathology were considered “pathological” (Table 2). For each patient, the diagnosis was provided by a physician responsible for the case. From the majority of donors (normal or patients), surgical specimens of bone with marrow were obtained. In the remaining cases, 1.4-mm bone core biopsies, together with aspirates, were obtained using a Jamshidi needle; for CFE experiments, only marrow from the core was used, while the aspirates were excluded to avoid variable degrees of peripheral blood contamination.

Cell suspensions

Bone marrow single-cell suspensions were prepared as described previously (Kuznetsov and Gehron Robey, 1996; Kuznetsov et al., 1997a, 1997b). Briefly, mouse and guinea pig bone marrow was flushed from the medullary cavities with α-modified minimum essential medium (αMEM; Invitrogen, Grand Island, NY, USA). Fragments of human trabecular bone and marrow were scraped with a steel blade into αMEM. The marrow preparations were then pipetted repeatedly to suspend the marrow cells and consecutively passed through 16- and 20-gauge needles to break up cell aggregates. The resulting cell suspensions were filtered through a 2350 nylon cell strainer (Becton–Dickinson, Franklin Lakes, NJ, USA) to remove the remaining cell aggregates.

Colony formation assay in primary marrow cell cultures

For the BMSC colony formation assay, marrow single-cell suspensions were plated in either triplicate or quadruplicate into 25-cm2 filter cap tissue culture flasks (Nalge Nunc International, Naperville, IL, USA) containing 5 ml of medium at the following densities: for mouse, 10 × 105, and for normal human specimens, 1–2 × 105 nucleated cells per flask. These cell numbers were selected based on the results of previous experiments such that discrete BMSC colonies would be formed in numbers sufficient for statistical analysis. In pathological human cases in which abnormal CFE could be expected, additional culture groups were prepared, with both lower (1 × 104) and higher (1 × 106) marrow cell numbers per flask.

Growth medium consisted of αMEM, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate (Invitrogen), and 20% FBS. The experiments comparing CFE in normal and pathological specimens of mouse and human marrow were carried out from 1994 to 2004 and employed the best FBS non-heat-inactivated lots available at the time (Invitrogen; Atlanta Biologicals, Inc., Norcross, GA, USA; Equitech-Bio, Kerville, TX, USA). A new lot was always chosen based on its ability to support both mouse and human CFE values comparable to the previous selected lot (see Figs. 1A and 1B, solid bars, as an example of how testing was conducted).

In experiments comparing the effects of noninactivated and heat-inactivated (56°C, 40 min) sera, FBS from 11 lots manufactured by five companies (Atlanta Biologicals; Biofluids, Inc., Rockville, MD, USA; Equitech-Bio; Gemini Bioproducts, Inc., Woodland, CA, USA; HyClone Laboratories, Inc., Logan, UT, USA) was used.

Mouse and human CFE cultures

In addition to FBS, mouse CFU-Fs (plated at low density) require extra stimuli provided by irradiated marrow cells to achieve optimal colony formation, while human CFU-Fs form the maximum number of colonies in just FBS-containing medium (Friedenstein et al., 1992; Kuznetsov and Gehron Robey, 1996; Latsinik et al., 1990). Consequently, mouse and human assays were performed differently. In mouse cultures, the whole marrow cell population was incubated for 2–4 h, the time sufficient for CFU-Fs to adhere to plastic (Castro-Malaspina et al., 1980; Latsinik and Epikhina, 1973). Then, nonadherent cells were aspirated, the cultures were washed with αMEM, and fresh growth medium was added. At this step, freshly prepared guinea pig bone marrow cells, γ-irradiated with 6000 R to prevent their proliferation (Friedenstein et al., 1992; Kuznetsov and Gehron Robey, 1996), were added at 1.0 × 107 nucleated cells per flask. In each mouse experiment, two or three extra flasks containing just irradiated guinea pig cells were prepared to ensure that these cells form no colonies on their own. No further medium replacements were performed thereafter. In human cultures, the entire population of plated cells was left undisturbed until harvest.

Cultivation and harvest

Cultivation was performed at 37°C in a humidified mixture of 5% CO2 with air. On day 9 to 12, the cultures were washed with Hanks' balanced salt solution (Invitrogen), fixed with absolute methanol, and stained with a saturated aqueous solution of methyl violet (Sigma, St. Louis, MO, USA). BMSC colonies containing 50 or more cells were counted using a dissecting microscope, and CFE (number of colonies per 1 × 105 nucleated marrow cells) was calculated.

Statistical analysis

Regression analysis was done using Microsoft Excel (Microsoft, Redmond, WA, USA). Unpaired t test, Welch corrected, or analysis of variance and posttest comparison using the Bonferroni multiple comparison test (InStat, GraphPad, San Diego, CA, USA) was performed. Differences were considered statistically significant at P < 0.05. In all figures, each bar represents the mean + SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Acknowledgments

The authors are grateful to Drs. Michael Collins (NIDCR, NIH); Jose Luis Franco and Steven M. Holland (NIAID, NIH); Glen Nuckolls (NIAMS, NIH); Clair A. Francomano (NIA, NIH); Elif Arioglu Oral and Philip Gorden (NIDDK, NIH); William Gahl (NHGRI, NIH); Arabella Leet, Paul Sponseller, Neal Fedarko, and Iain McIntosh (Johns Hopkins University, Baltimore, MD); John W.T. Walker and Stephen L.-K. Yen (Children's Hospital, Los Angeles, CA); Fred Singer (John Wayne Cancer Institute, Santa Monica, CA); Alan Aaron and Brian Evans (Georgetown University, Washington, DC); Paul A. Manner (George Washington University, Washington, DC); and Michael Keating (Children's National Medicine Center, Washington, DC) for providing human bone and bone marrow. We are indebted to Drs. Kenn Holmbeck (NIDCR, NIH); Tomoko Iwata and Clair A. Francomano (NIA, NIH); Cui-Ling Li and Chu-Xia Deng (NIDDK, NIH); Lorraine A. Fitzpatrick (GlaxoSmithKline, Collegeville, PA); and Laura Calvi, Henry M. Kronenberg, and Ernestina Schipani (Massachusetts General Hospital and Harvard Medical School, Boston, MA) for providing transgenic mice with skeletal phenotypes. This research was supported by the Division of Intramural Research of the National Institute of Dental and Craniofacial Research, IRP, NIH, DHHS.

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