Role of muscle-derived cells in hematopoietic reconstitution of irradiated mice

Experimental animals: Female and male Kunming mice were obtained from the Department of Experimental Animals, Institute of Hematology, Chinese Academy of Medical Sciences. Weight of female mice: 35g, weight of male mice: 20g.

Culture of muscle cells: Male Kunming mice were sacrificed and muscle tissue was removed from the femurs under sterile conditions. The muscles were cut into small cubes and transferred to 10 ml test tubes for incubation in 4% trypsin-Hanks balanced salt solution at 37°C for 10 minutes. A 5 ml aliquot was then transferred to a fresh tube, and 5 ml RMPI 640 culture solution was added to stop the trypsin reaction. An additional 5 ml of 4% trypsin was added to the first tube to continue the reaction. After repeating this process 5-6 times, the collected cells were centrifuged and resuspended in RPMI 1640 with 10% serum.

Muscle-derived cell rescue of irradiated animals: To determine the ability of muscle-derived cells to rescue lethally irradiated mice, 75 Kunming female mice were irradiated with 8.5 Gy by Gamma cell 40 (cerium) and then divided into 5 groups of 15 mice each. Group 1: irradiation only. Group 2: injected through tail vein on day 2 after irradiation with 0.2 ml muscle cell culture medium. Group 3: injected on day of irradiation with 0.2 ml muscle cells (10,000 cells/ml). Group 4: injected on day 2 after irradiation with 0.2 ml muscle cells (10,000 cells/ml). Group 5: injected on day 2 after irradiation with 0.2 ml muscle cells (10,000 cells/ml) that had been cocultured with BMP2 for 24 hours. The survival of these animals was then compared with animals rescued with BM as described below. The results of these studies were used to determine the minimum number of muscle-derived cells needed for rescue in the engraftment studies described below.

Bone marrow rescue of irradiated animals: BM and PB cells were obtained from male Kunming mice, and mononuclear cells were isolated by Ficoll centrifugation. Sixty female Kunming mice were irradiated with 8.5 Gy by Gamma cell 40 (cerium) and divided into 4 groups of 15 mice each. Group 1: injected on day 2 after irradiation with 0.2 ml PB cells (10,000 cells/ml). Group 2: injected on day 2 after irradiation with 0.2 ml BM cells (10,000 cells/ml). Group 3: injected on day 2 after irradiation with 0.2 ml BM cells (100,000 cells/ml). Group 4: injected on day 2 after irradiation with 0.2 ml BM cells (100,000 cells/ml) incubated with BMP2 for 24 hours.

Pathology studies: The femurs and spleens of all dead animals were fixed in 10% formaldehyde, sectioned, and stained with hematoxylin and eosin for histological studies.

Source of engrafted cells in animals rescued with muscle-derived cells: To determine the source of engrafted cells in animals rescued by injection of muscle-derived cells, 60 female Kunming mice were divided into two groups of 30 mice each. The animals were irradiated as described above. The first group did not receive additional cells for rescue. The second group was injected with 0.2 ml of muscle-derived (10,000 cells/ml) as described above. Threemice from each group were evaluated every 3 days by C band chromosome analysis and Y chromosome polymerase chain reaction (PCR) (see below).

C band chromosome analysis: BM cells were harvested from sacrificed mice, cultured at 37°C for 2-3 weeks, and then incubated with colchine. C-band analysis was then performed.

Y-chromosome polymerase chain reaction: PCR was performed using BMcells obtained from all expired female mice to determine if any male-derived cells were present. Processed DNA samples were amplified in 50ul containing 20 pmols of mouse Y chromosome-specific primers and mouse PDGF B receptor-specific primers,1 as well as 1.5mM MgCl, 50 mM KCl, 10 mM Tris-Hcl, pH 8.3, 0.2 mM dNTPs, and 1.5 units Taq polymerase (SABC, China). Y-chromosome specific primers: (sense primer) CTG CTG TGA ACA GAC ACT AC; (anti-sense primer) GAC TCC TCT GAC TTC ACT TG. Mouse PDGF B receptor primers: (sense primer) CAT TGG CTC CAT CCT GCA TA; (anti-sense primer) GGA TAA GCC TCG AAC ACC AC. PCR was initiated at 94°C for 4 minutes, followed by 30 cycles of 94°C for 1 minute, 62°C for 1 minute, and 72°C for 2 minutes, followed by a final cycle of 72°C for 5 minutes. Amplification with Y-chromosome specific primers results in a 722 bp fragment corresponding to the srylocus sequence 256-978. Amplification with PDGF B receptor primers generates a fragment of approximately 750 bp corresponding to the mouse PDGF B receptor cDNA sequence 948-1166, which contains an intron. All PCR amplifications included a male (positive) and female (negative) control.

Rescue of irradiated mice: In the first set of experiments in which muscle-derived cells were injected into irradiated mice, all animals from Groups 1, 2, and 5 died between days 9-13 after irradiation. In contrast, 3/15 mice from Group 3 and 8/15 mice from Group 4 have survived for over 2 months (Table1). In the second set of experiments in which peripheral blood or BM cells were injected into irradiated mice, all animals from Groups 1 and 2 died between days 9 -13 after irradiation. In contrast, 12/15 mice from Group 3 and 13/15 mice from Group 4 have survived (Table 2). Bone marrow and spleen sections were obtained from all dead mice from both sets of experiments. All animals studied exhibited empty BMs and splenic atrophy. No differences were seen among the different study groups. In addition, Y-chromosome specific PCR was performed on BM tissue obtained from the dead mice. No male-derived cells were detected in the BMs of any dead mice.

Source of engrafted cells in animals rescued with muscle-derived cells: On day 3 after irradiation, only female-derived cells were present in BM by C-band chromosome analysis. On day 9, 1.7% of cells were of male origin. From days 12-18, in general, only male-derived cells were present, with only a few female-derived cells found . On day 21, female-derived cells were again present. In the irradiation-only control group, male-derived cells were never found. Similarly, PCR amplification of the Y-chromosome specificsry gene revealed the presence of male-derived cells in BM from day 9 after irradiation and beyond.

Hematopoietic stem cells are thought to have the property of self-renewal, and to be able to differentiate into a variety of hematopoietic lineages. Previous studies have demonstrated that they have the capacity to differentiate but have been unable to demonstrate self-renewal. One difficulty has been how to identify a hematopoietic stem cell, becauseby the time a cell is defined as such, it has already differentiated into a committed progenitor.

We found that hematopoiesis can be reconstituted in lethally irradiated mice by infusion of muscle-derived cells. This demonstrates that some muscle-derived cells have the ability to differentiate into hematopoietic cells. The addition of BMP2 blocks the ability of these cells to reconstitute hematopoiesis. This suggests that these cells express receptors for BMP2, and that BMP2 induces these muscle-derived cells to differentiate into bone rather than hematopoietic tissue2,3. Previous studies have demonstrated that BMP2 can induce muscle cells to differentiate into bone both in vivo and in vitro. It is possible that these muscle-derived cells can function as stem cells for both hematopoietic and bone tissue. One outstanding question is whether muscle tissue is heterogeneous in nature and contains several types of stem cells for different tissues. Alternatively, muscle-derived cells may be homogeneous in nature, and retain the capacity to differentiate into other tissue types under the appropriate conditions.

We found that administration of BM or PB cells could not save lethally irradiated mice when administered at the same concentration (10,000 cells/ml) as muscle-derived cells. Rather, a tenfold increase in BM cells was needed for hematopoietic rescue. This result shows that muscle-derived cells are more efficient in reconstituting hematopoiesis than BM cells. In addition, BMP2 did not block BM cells from rescuing the irradiated mice. Therefore, muscle-derived cells differ from BM cells. They are not blood cells, but can differentiate into hematopoietic tissue.

On day 9 after irradiation, male-positive blood cells were present in the BM of rescued mice. This demonstrates that muscle-derived cells were able to reconstitute hematopoiesis. However, some mice died despite being treated in the same way. This shows that while only a few muscle-derived cells are needed to reconstitute hematopoiesis, the cell number we used was lower than that needed to rescue all the experimental animals. Additional experiments are needed to identify the optimal cell number for rescue. The above studies illustrate that muscle-derived cells provide a highly efficient source of cells that are able to differentiate into hematopoetic tissue. In addition, selection of these cells in vitro for transplant may be a better way to obtain hematopoietic stem cells than BM because muscle tissue is the biggest tissue in the body, and a donor is not needed.

On day 21 after irradiation, female-positive blood cells were present in the BM of rescued mice. This demonstrates that the irradiated mice could renew hematopoiesis by themselves. The time needed for reconstitution of hematopoiesis in this manner was longer than that required for reconstitution by the transplanted cells. It is not known which cells are able to reconstitute hematopoiesis, and why more time would be needed for this process to occur. If there are hematopoietic stem cells circulating in the blood of the treated mice, then they should be able to reconstitute hematopoiesis in a shorter period of time than the transplanted cells. We hypothesize that there are no true hematopoietic stem cells circulating in the blood, but rather only progenitors. The true stem cells appear to be the common ancestors of other tissues such as bone and blood, which need time to enter BM and to differentiate into blood cells.

The above studies demonstrate that some muscle-derived cells can differentiate into blood cells, and that these cells express receptors for BMP2 on their surface. Further purification of these cells would provide a new way to perform bone marrow transplant, and to resolve the difficulty of in vivo proliferation of hematopoietic stem cells.