The effect of recombinant human bone morphogenetic protein-2 on untreated and CD34-selected umbilical cord blood.
The bone morphogenetic proteins or BMP's comprise a growing family of more than fifteen proteins. The term BMP was coined by Marshall R. Urist nearly 30 years ago after observing heterotopic or extraskeletal bone formation after implantation of demineralized bone matrix at intramuscular sites in rodents and rabbits (Urist, 1965). It was Urist' theory of autoinduction which brought the term "bone morphogenetic protein" to light. He found that an induction cell such as a histiocyte acts upon a connective tissue cell facilitating differentiation into the osteoprogenitor cells. This bone inducing substance was referred to as bone morphogenetic protein (Lee, 1997). Since that time many researchers have attempted to characterize and clone these proteins for further study.
Human BMP-2 is a 32-kd homodimeric glycoprotein. The receptors for BMP-2 contain a serine/threonine kinase domain and has been successfully cloned by Wozney et al, 1988. The recombinant BMP-2 has proliferative effects on the multipotential stem cells toward the fibroblastic, osteoblast/osteocyte, and adipocyte cell lines (Yamaguchi et al, 1991 and Sigurdsson et al, 1995).
The goal of this research was to determine if recombinant BMP-2 had a proliferative effect on hematopoietic stem cells isolated from umbilical cord blood. It is well documented umbilical cord blood is a rich source of hematopoietic stem cells (Hughes, 2000 and Cardoso et al, 1993). Numerous studies have demonstrated recombinant BMP-2's effect on the bone forming cells and fibroblasts. The fact that osteoblasts secrete two major cytokines, namely, macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) led this group to believe that recombinant BMP-2 may play a major role in hematopoiesis, and further, serve as a potential cytokine in stem cell transplantation.
MATERIALS AND METHODS
Umbilical cord blood was collected into sterile 50 mL conical tubes containing 10% (ACD-A) as a preservative. Cord blood was collected from the University of Maryland Medical Center, Department of Obstetrics and Gynecology and approved by the Institutional Review Board of the University of Maryland.
Fifteen milliliters of cord blood was diluted to 45 mL with PBS (1x) in a 50 mL conical tube. Five mL of filtered 10% dextran (mol wt. 250,000) was added to the conical tube. The tube was incubated for 30 minutes at room temperature and the supernatant was centrifuged at 400 g for 10 minutes to recover mononuclear cells. The resulting pellet was washed with 5% Bovine Serum Albumin (BSA) in PBS (Phosphate buffered saline).
Mononuclear cells were isolated for stem cell content via purification flasks from Applied Immune Sciences, Inc (Santa Clara, CA). Cells were first incubated with Soybean Agglutinin (SBA) flasks to reduce the number of T cells and fibroblasts. SBA binds to galactose present on these cells but will not bind to stem cells. The non-adherent cells were then incubated with flasks containing antibody to CD34.
AIS MicroCELLector[TM] SBA
MicroCELLector flasks were primed with 10 mL of sterile DPBS (Dulbecco's phosphate buffered saline) and incubated for 1 hour at room temperature. After incubation residual DPBS was removed and flasks were washed an additional four times with DPBS. Cord blood cells were resuspended into DPBS containing 0.5% human gamma globulins and incubated for 15 minutes at room temperature. Mononuclear cells were loaded and incubated for 1 hour at room temperature. After incubation the non-adherent portion was collected. An additional 4 mL of DPBS was added to the flask to collect any additional cells not adhering to the flask. The SBA-neg cells were centrifuged for 5 minutes at 840 g at room temperature. The cell pellet was resuspended in DPBS containing 0.5% human gamma globulins. Viability was determined by Trypan Blue Exclusion.
AIS MicroCELLector[TM] CD34 Isolation
CD34 flasks were prepared as previously described using SBA flasks. The SBA-negative fraction was incubated in flasks containing antibody to CD34 at no more than 2 X 1[0.sup.7] cells/flask for 1 hour at room temperature. At the end of the 1 hour incubation the flask was rocked from side to side and the non-adherent population was removed with a sterile pipet. Four mL of DPBS containing 0.5% BSA was added to the flask and rocked from side to side. Residual non-adherent cells were again removed. An additional aliquot of DPBS containing 0.5% BSA was added to the flask. With the cap tightly bound, the narrow side of the flask was hit firmly to disrupt the adherent CD34 population. The adherent cells were removed with a sterile pipet. The adherent suspension was centrifuged at 100 g for 20 minutes at room temperature and a cell count was determined.
Clonogenic Assays: CFU-GM
Methylcellulose semi-solid media (Stem Cell Technologies, Inc, Vancouver, BC) containing the following growth factors: stem cell factor (50 ng/mL), GM-CSF (10 ng/mL), Interleukin-3 (10 ng/mL), and Erythropoietin (3 U/mL). Cord blood cells from the untreated and CD34-selected populations were plated according to the following formula: plating density/cell count. Recombinant BMP-2 was added at concentrations of 25 and 100 ng/mL. Media/cell mixture was plated in duplicate into two 35 X 100 mm sterile petri dishes. A third dish contained 3 mL of sterile water to serve as a humidifier. The three petri dishes were placed into a larger 15 X 100 mm tissue culture plate and incubated at 37 C with 5% C[O.sub.2] for 2 weeks. Colonies of CFU-GM (Colony forming unit-granulocyte macrophage; Fig. 1) were scored after 2 weeks incubation using an inverted microscope.
[FIGURE 1 OMITTED]
Recombinant BMP-2 was acquired from the Genetics Institute (Cambridge, MA) at a concentration of 2.27 mg/mL and stored at -80[degrees] C. Dilutions were performed using sterile 0.1% BSA in PBS and concentrations of 100 ng/mL and 25 ng/mL were aliquoted into siliconized tubes and stored at -80[degrees] C.
A one-way analysis of variance (ANOVA) was utilized to test the significance of recombinant BMP-2 at concentrations of 0, 25, and 100 ng/mL added to the untreated and CD34-selected cord blood.
The response of CFU-GM to recombinant BMP-2 at concentrations of 0, 25, and 100 ng/mL from untreated cord blood is depicted in Figure 2. In the absence of recombinant BMP-2 the CFU-GM ranged from 3.0 X 1[0.sup.3] to 8.8 X 1[0.sup.3]/mL with an average of 5.7 X 1[0.sup.3]mL. In the presence of 25 ng/mL of growth factor the range was identical to cultures devoid of recombinant BMP-2. In the presence of 100 ng/mL of growth factor the range of CFU-GM was 1.0 X 1[0.sup.3] to 3.3 X 1[0.sup.4]/mL, with an average of 1.3 X 1[0.sup.4]/mL. While the average of CFU-GM was largest in cultures containing the highest concentration of recombinant BMP-2 ANOVA yielded a P>.05.
The CD34-selected cord blood clonogenic assays with recombinant BMP-2 is depicted in Figure 3. In the absence of growth factor the numbers of CFU-GM ranged from 20 to 420 mL with an average of 230 colonies/mL. In the presence of 25 ng/mL the range of CFU-GM was 10 to 420/mL with an average of 230 colonies/mL. In the presence of 100 ng/mL of recombinant BMP-2 the range of CFU-GM was 20 to 360/mL with an average of 216 colonies/mL.
The viability determination by trypan blue exclusion methodology yielded greater than 90% viable cells from the untreated cord blood population, range 96 to 100%. In the CD34-selected population viability was also greater than 90% in all samples with a range of 92 to 98%.
This study sought to test the effects of recombinant BMP-2 on two populations of hematopoietic cells from umbilical cord blood. It has been well documented that this growth factor stimulates local progenitor cells to differentiate into chondrocytes (cartilage forming cells) and osteoblasts and thus play an important role in skeletal development (Woo, 2001). In addition, fibroblasts which are an integral component of the bone marrow where hematopoiesis takes place secrete GM-CSF, G-CSF (Granulocyte-colony stimulating factor), CSF-1 (Colony stimulating factor-1), and IL-6 (Interleukin-6). Osteoblasts secrete IL-6 and GM-CSF. The precursor of osteoclasts is thought to be the monocyte, therefore, osteoblasts indirectly provide the growth factor for synthesis of the osteoblasts, which is CSF-1. It was therefore hypothesized that recombinant BMP-2 would likewise have a proliferative effect on the hematopoietic cells.
A total of five cord blood samples were collected from the umbilical cord at birth. Cultures were plated containing methylcellulose semi-solid media to which nucleated cells from the untreated and CD34-selected populations were added. Concentrations of growth factor were added at 0, 25, and 100 ng/mL in duplicate. There was no significant difference between the control group and in cultures seeded with recombinant BMP-2 using one-way ANOVA. The studies utilizing recombinant BMP-2 as a growth factor are few in number. Bhatia et al, 1999 observed differential effects to the primitive CD34+CD38- cell at a concentration of recombinant BMP-2 of 5 ng/mL. At higher concentrations (50 ng/mL) differentiation was inhibited. Recombinant BMP-4, a member of the BMP superfamily was also tested on primitive cells. This growth factor appeared to mimic responses of recombinant BMP-2 at 5 ng/mL and inhibited concentrations at higher concentrations (Li et al, 2001).
Ploemacher et al, 1999 tested recombinant BMP-9 at a concentration of 3 ng/mL on murine bone marrow cells and found an increased number of colony forming cells in semisolid cultures compared to higher concentrations. Cytokines are collectively characterized as being synergistic in nature and future studies should consider utilizing this characteristic with recombinant BMP-2, 4, and 9 at low concentrations.
Viability studies were performed via trypan blue exclusion to ensure live cells were cultured. Viability for all samples was greater than 90%. This percentage is consistent with previous studies involving cord blood. (9)
In conclusion, this study established that recombinant BMP-2 had no significant effect on hematopoietic clonogenic assays for CFU-GM at seeded concentrations of 25 and 100 ng/mL. Subsequent experiments should explore the synergistic effects of other BMP's with recombinant BMP-2 at low concentrations on hematopoietic cells.
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Virginia C. Hughes
School of Sciences
Medical Technology/Clinical Lab Science
Auburn University Montgomery
Montgomery, AL 36124
Denise M. Harmening
Department of Medical and Research Technology
University of Maryland, School of Medicine
Baltimore, Maryland 21201