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Journal of Clinical Oncology, Vol 18, Issue 2 (January), 2000: 307
© 2000 American Society for Clinical Oncology

Rapid Hematopoietic Recovery After Coinfusion of Autologous-Blood Stem Cells and Culture-Expanded Marrow Mesenchymal Stem Cells in Advanced Breast Cancer Patients Receiving High-Dose Chemotherapy

By Omer N. Koç, Stanton L. Gerson, Brenda W. Cooper, Stephanie M. Dyhouse, Stephen E. Haynesworth, Arnold I. Caplan, Hillard M. Lazarus

From the Departments of Medicine and Biology, Case Western Reserve University; and Division of Hematology/Oncology and Ireland Cancer Center of University Hospitals of Cleveland, Cleveland, OH.

Address reprint requests to Omer N. Koç, MD, Case Western Reserve University, BRB-3 Hematology/Oncology, 10900 Euclid Ave, Cleveland OH 44106; email onk2{at}po.cwru.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion.

PATIENTS AND METHODS: We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor.

RESULTS: Human MSCs were successfully isolated from a mean ± SD of 23.4 ± 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 106 MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 106 MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 106 expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/µL and platelet count >= 20,000/µL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively.

CONCLUSION: This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUMAN BONE MARROW contains mesenchymal progenitors (mesenchymal stem cells [MSCs]) that produce adventitial cells in the marrow microenvironment; these cells provide support to hematopoiesis by producing membrane-bound and soluble signals and cytokines. These stromal progenitors can be readily isolated from bone marrow and demonstrate extensive proliferative capacity in vitro.1 Purified and culture-expanded human MSCs differentiate along the osteogenic,2 chondrogenic, and adipogenic lineages3 both in vitro and in vivo. In unstimulated cultures, MSCs appear as fusiform fibroblasts with expression of unique surface proteins (SH2, SH3, SH4) that are not found on hematopoietic precursors.3 MSCs lack expression of hematopoietic markers such as CD45, CD14, and CD34.3,4 MSCs constitutively secrete interleukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14, IL-15, macrophage colony-stimulating factor, Flt-3 ligand, and stem-cell factor, and they are inducible with IL-1{alpha} to produce IL-1{alpha}, leukemia-inhibiting factor, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor.5 Similar to Dexter-type stromal cultures, MSCs can support human long-term culture-initiating cells (LTC-ICs). Therefore, we postulated that MSCs may enhance hematopoietic engraftment rate and quality after myeloablative and stroma-damaging treatments.

In a pilot study, our group demonstrated the safety of ex vivo expansion and subsequent infusion of autologous MSCs in 15 patient volunteers.6 These individuals had hematologic malignancies that were in remission at the time of MSC collection and infusion and were not given preparative chemotherapy. Only 1 to 50 x 106 total autologous MSCs were administered via intravenous (IV) infusion without any toxicity. However, human bone marrow–derived, culture-expanded MSCs have never been administered via IV infusion into patients at the time of peripheral-blood progenitor-cell (PBPC) transplantation with a therapeutic intent. We now report results of a phase I-II clinical trial performed to determine the feasibility, safety, and hematopoietic effects of bone marrow–derived culture-expanded autologous MSCs infused into patients in the course of high-dose chemotherapy and hematopoietic stem-cell rescue. Our results show that autologous MSCs can be successfully culture-expanded and infused along with PBPCs after high-dose chemotherapy in advanced breast cancer patients, are free of toxicity, and are associated with rapid hematopoietic recovery.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Between October 1996 and July 1998, 32 patients with locally advanced or metastatic breast cancer who were eligible for high-dose chemotherapy and PBPC transplantation were enrolled onto this phase I-II trial at the Ireland Cancer Center, University Hospitals of Cleveland, Case Western Reserve University in Cleveland, OH, after obtaining written informed consent. The clinical trial protocol and the consent form were approved by the Institutional Review Board for Human Investigation of the University Hospitals of Cleveland. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0 or 1 and were required to have adequate visceral organ function, including a left ventricular ejection fraction of at least 50%, forced expiratory volume in 1 second and diffusion capacity of carbon monoxide greater than 50% predicted, serum direct bilirubin less than 2.0 mg/dL, and an actual or calculated creatinine clearance greater than 60 mL/min. At the start of therapy, a neutrophil count greater than 1.2 x 109/L and a platelet count greater than 100 x 109/L were required. Patients were excluded if they had cumulative doxorubicin exposure in excess of 500 mg/m2, major CNS dysfunction, active infection, or a history of autoimmune disease. Patients were not excluded for evidence of tumor on routine histologic staining of bilateral paraffin-embedded posterior iliac crest bone marrow biopsy specimens.

Ex Vivo MSC Culture
On enrollment, approximately 35 days before scheduled PBPC infusion (see High-Dose Chemotherapy and PBPC Infusion), 20 to 25 mL of bone marrow aspirate was obtained under sterile conditions by puncture of bilateral posterior iliac crests of patients under local anesthesia. Aspirates were obtained 2 to 48 hours before high-dose cyclophosphamide mobilization chemotherapy. The aspirate was taken to the class 10,000 quality clean production suite of the Cell and Gene Therapy Core Facility at the Case Western Reserve University. Aspirate was mixed with two volumes of Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY) in a sterile class II biologic safety cabinet and centrifuged at 900 x g for 10 minutes at 20°C in a Beckman GS-6R centrifuge (Palo Alto, CA). Pellets were layered onto 25 mL of Percoll (density, 1.073 g/mL) (Sigma, St Louis, MO) at a density of 1 to 2 x 107 cells/mL. Gradients were centrifuged at 900 x g for 30 minutes at 20°C, and recovered mononuclear cells were resuspended in DPBS and centrifuged at 460 x g for 10 minutes at 20°C. Cells were resuspended at 1 x 106 cells/mL in Dulbecco’s modified Eagle medium, low glucose (DMEM-LG) (GibcoBRL, Grand Island, NY) with 10% fetal bovine serum (Hyclone, Logan, UT,) and 30 mL of cell suspension was plated in a 175 cm2 flask (Falcon, Franklin Lakes, NJ). The serum lot used was selected on the basis of optimal MSC growth with maximal retention of osteogenic differentiation as assessed with in vitro and in vivo assays.7 MSCs were cultured in humidified incubators with 5% CO2 and initially allowed to adhere for 72 hours, followed by media change every 3 to 4 days. When cultures reached more than 90% confluence, adherent cells were detached with 0.05% trypsin-EDTA (Gibco) and replated (passaged) at a density of 1 x 106 per 175 cm2 flask until processing for infusion. Cell cultures were tested for sterility weekly (University Hospitals Microbiology, Cleveland, OH) and for the presence of breast cancer cells by immunocytochemical method (BIS Labs, Reseda, CA),8 endotoxin by limulus amebocyte lysate test (Associates of Cape Cod, Falmouth, MA), and Mycoplasma by DNA-fluorochrome stain (Bionique, Saranac Lake, NY) before infusion.

Flow Cytometry
Surface expression of SH-2, SH-3, SH-4, CD14, and CD-45 was determined on culture-expanded MSCs. Cells were detached with 0.05% trypsin-EDTA (Gibco), washed with DPBS plus 2% bovine albumin, fixed in 1% paraformaldehyde, blocked in 10% normal goat serum, and incubated separately with primary SH-2, SH-3, and SH-4 antibodies (Osiris Therapeutics, Baltimore, MD) followed by phycoerythrin-conjugated antimouse IgG(H+L) antibody (Caltag, Burlingame, CA) or with fluorescein isothiocyanate conjugated CD45 and phycoerythrin-labeled CD14 with appropriate isotype controls (Becton Dickinson, San Jose, CA). Flow cytometry was performed on a FACScan (Becton Dickinson, Parsippany, NJ) equipped with an argon laser, and data were analyzed with CellQuest software (Becton Dickinson).

High-Dose Chemotherapy and PBPC Infusion
The PBPC mobilization regimen consisted of cyclophosphamide 4.0 g/m2 IV infusion over 6 hours on day 1, along with mesna (first 3.0 g/m2 IV, then 500 mg every 3 hours orally/IV for eight doses) and prednisone 2.0 mg/kg orally on days 1 through 4.9 At 36 to 48 hours after completion of the cyclophosphamide, patients began subcutaneous injections of recombinant human G-CSF (Amgen, Thousand Oaks, CA) 10 µg/kg/d. On recovery of neutrophils to greater than 1 x 109/L (usually 12 to 15 days after cyclophosphamide treatment) patients underwent a leukopheresis procedure using Cobe Spectra (COBE, Lakewood, CO) pheresis equipment. A pheresis with a mean volume of 20 L (4 x total blood volume) was performed in each session, which was repeated until >= 2.0 x 106 CD34+ cells/kg or >= 12 x 108 mononuclear cells/kg of patient weight was obtained. Cells were cryopreserved using a controlled-rate liquid nitrogen freezer using previously published methods.10 After PBPC procurement, high-dose chemotherapy with cyclophosphamide 6,000 mg/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 were administered as continuous IV infusion over 96 hours through a central venous catheter from days T-7 through T-3.11 PBPCs were thawed and infused 72 hours after the completion of high-dose chemotherapy (day T-0). All patients received recombinant human G-CSF 10 µg/kg subcutaneously (Amgen) daily starting 4 hours after the PBPC infusion until neutrophil engraftment (absolute neutrophil count > 0.5 x 109/L for 3 days). Platelet engraftment was defined as the first of 7 consecutive days on which platelet count was greater than 20 x 109/L without transfusion support. Bone marrow aspirates were obtained on days T+7, T+14, T+42, and T+72 to determine hematopoietic colony-forming unit (CFU) recovery. Patients underwent restaging evaluation with computed tomography and bone scans 42 days after transplantation and every 3 months thereafter.

Supportive Care
All patients had multilumen, indwelling central venous pheresis catheters and were cared for in single hospital rooms. Antibiotics were given empirically for fever and neutropenia, and all patients were supported with irradiated blood components. Irradiated, packed RBC transfusions were given in an attempt to keep the hematocrit greater than 25%, and irradiated platelet transfusions were given for platelet counts less than 10 x 109/L or bleeding complications. cytomegalovirus-negative blood products were given to cytomegalovirus-seronegative patients. Toxicity grading was accomplished using the National Cancer Institute common toxicity criteria.

MSC Infusion
Confluent MSCs in 175 cm2 flasks (15 to 85 per patient) were washed with Tyrode’s salt solution (Sigma), incubated with Medium 199 (M199 [Gibco]) for 60 minutes, and detached with 0.05% trypsin-EDTA (Gibco). Cells from 10 flasks were detached at a time and MSCs were resuspended in 40 mL of M199 plus 1% human serum albumin (HSA [American Red Cross, Washington, DC]). MSCs harvested from each 10-flask set were stored for up to 4 hours at 4°C and combined at the end of the harvest. A total of 1 to 2.2 x 106 MSCs/kg were combined and resuspended in M199 + 1% HSA and centrifuged at 460 x g for 10 minutes at 20°C. Cell pellets were resuspended in fresh M199 + 1% HSA media and centrifuged at 460 x g for 10 minutes at 20°C for three additional times. MSCs were then resuspended in M199 + 1% HSA at 1 x 106 cells/mL and transferred into 20-mL syringes. Total harvest time was 3 to 5 hours on the basis of MSC yield per flask and the target dose. Freshly harvested autologous MSCs were infused into patients 1 or 24 hours after PBPC infusion (on day T-0 or T+1) through a side port of a running 0.9% saline IV infusion into a central catheter over 15 to 20 minutes. Patients were premedicated with acetaminophen 650 mg and diphenhydramine 25 to 50 mg. The protocol was amended in March 1998 to allow cryopreservation of the harvested MSCs and to increase the cell dose to 2 x 106 MSCs/kg. In eight patients, harvested MSCs were cryopreserved with a rate-controlled freezer in a final concentration of 10% dimethyl sulfoxide (Research Industries, Salt Lake City, UT) and 5% autologous plasma in Cryocyte (Baxter, Deerfield, IL) freezing bags. Bags were overfilled with >= 10% MSCs to account for cell loss during freeze and thaw. On the day of infusion cryopreserved units were thawed at the bedside in a 37°C water bath and transferred into 60-mL syringes within 10 minutes and infused into patients as described above. Vital and clinical signs and symptoms were monitored at the time of infusion and every 15 minutes thereafter for 3 hours, followed by every 2 hours for 6 hours and every 8 hours for 3 days.

Detection of MSCs in Blood
Approximately 10 to 15 mL of peripheral-blood samples were collected before MSC infusion, immediately at the end of infusion (0 minutes), and 15 minutes and 60 minutes after infusion. RBCs were lysed with ammonium chloride buffer, and cells were washed with DPBS and plated in 35-mm plates with a 1:1 mixture of fresh DMEM-LG and culture-conditioned and filtered DMEM-LG. Media was changed twice a week starting at 72 hours. Cultures were scored positive if a layer of adherent cells (>90% confluent) with MSC morphology and surface expression of SH3 were detected.

Hematopoietic CFU Assay
Bone marrow mononuclear cells were grown in methylcellulose (Stem Cell Technologies, Vancouver, BC) containing a final concentration of 100 U/mL hIL-3 (Sandoz Research Institute, Nutley, NJ), 2 U/mL erythropoietin (Amgen), 100 U/mL granulocyte-macrophage colony-stimulating factor (Immunex, Seattle, WA) and 0.1 mmol/L hemin (Sigma), as described previously.10 Cells were plated at a density of 1 x 105/mL in duplicate and grown at 37°C 5% CO2. Twelve to 14 days later, colonies of greater than 50 cells were enumerated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
The median age of the 32 enrolled patients was 47 years (range, 37 to 57 years) and the majority had stage IV breast cancer (Table 1). Four patients were taken off study. Two experienced disease progression after PBPC mobilization therapy and did not proceed to high-dose chemotherapy. Two other patients had breast cancer cells in their marrow aspirates that persisted in MSC cultures. These patients underwent high-dose chemotherapy and PBPC infusion but were not infused with MSCs.


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  		Table 1. Patient Characteristics
 
MSC Cultures
Human bone marrow–derived MSCs were successfully isolated from a mean of 23.4 ± 5.9 mL of bone marrow aspirate from all 32 breast cancer patients, and all were successfully culture-expanded (Table 2). A morphologically homogenous population of fibroblast-like MSCs was detected in primary cultures (Fig 1), which reached greater than 90% confluence in a median of 13 days (day of first passage). During late passages, MSCs had a larger appearance but remained homogenous. There was a good correlation (r = 0.753) between the number of mononuclear cells plated to initiate MSC cultures and the number of MSCs recovered at the first passage (Fig 2). A mean of 1.4 ± 0.7 x 105 MSCs were recovered at the first passage from 1 x 106 input bone marrow mononuclear cells. MSCs that were recovered from the first passage were replated at 1 x 106 MSCs per 175-cm2 flask. These cultures reached greater than 90% confluence in 7 days, which required weekly passages. MSC growth after the first passage was exponential, and the number of MSCs increased more than 2 logs for cultures maintained for six weekly passages (Fig 3). Mean MSC yield per 175-cm2 flask was 4.5 x 106 MSCs (range, 1.9 to 8.8 x 106 MSCs) at the first passage and 3.4 x 106 MSCs (range, 2.0 to 5.0 x 106 MSCs) at the third passage. Due to culture laboratory space limitations, only that portion of MSCs that were predicted to yield two to three times the target MSC dose was maintained in culture. Potential total MSC yield was calculated on the basis of the actual expansion of MSCs in the cultured portion of MSCs from each passage and extrapolated to the total number of MSCs obtained from that passage (Table 3). Potential total MSC yield per kilogram of patient weight increased from a mean of 5.6 x 106 MSCs/kg after two passages to a mean of 36.3 x 106 MSCs/kg after six passages. Although the majority of patients’ cultures contained at least the target number (1 x 106/kg) of MSCs by day 21, median culture duration was 37 days (range, 20 to 50 days) due to obligatory delay between mobilization of PBPCs and institution of the high-dose chemotherapy. This duration was reduced to a median of 28.5 days by implementation of MSC cryopreservation.


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  		Table 2. MSC Culture Data
 


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  		Fig 1. Phase contrast photomicrograph of cultured MSCs (magnification x100). (A) Single fusiform adherent cells early in the primary culture. (B) Late (day 13) in the primary culture, showing confluent patches of MSCs immediately before first passage. (C) Confluent layer of fifth-passage MSCs.

 


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  		Fig 2. Correlation between number of bone marrow mononuclear cells used to start MSC cultures and the number of MSCs obtained at the end of primary culture (first passage). Each filled circle represents an individual patient. Abbreviation: BM, bone marrow.

 


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  		Fig 3. Culture expansion of MSCs. Average number of MSCs per patient at each passage. Cultures were grouped on the basis of total passages (P): six Ps, n = 7; five Ps, n = 8; four Ps, n = 6; three Ps, n = 6; two Ps, n = 3. Total MSC yields were calculated on the basis of the portion expanded and total cells available for expansion. Error bars represent one SD from the mean.

 

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  		Table 3. Human MSC Expansion Potential
 
There was no significant correlation between either the number of MSCs obtained at the first passage or MSC expansion (as total estimated MSC yield divided by the number of passages) and the number of prior chemotherapy regimens (one prior chemotherapy regimen v two or more, P = .2 and .5, respectively) or prior radiotherapy treatments (one prior treatment v two or more, P = .7 and .6, respectively). In addition, we found no correlation between the number of CD34+ cells collected per leukopheresis and the number of MSCs harvested at first passage (r = 0.18). There was a weak correlation between the number of CD34+ cells collected per leukopheresis and the MSC expansion (as total estimated MSC yield, divided by the number of passages) (r = 0.379) and the number of MSCs per 175-cm2 flask at the first passage (r = 0.282).

There was no evidence of bacterial, fungal, or Mycoplasma contamination in any of the 3,029 flasks processed. Cell viability was determined by trypan blue staining at the end of the harvest and before infusion and was greater than 95% in every infusate at both time points. Cells were characterized by flow cytometry using human MSC-specific monoclonal antibodies that react with surface antigens of MSCs designated SH2, SH3, and SH4 before infusion. Every harvest revealed a homogenous population of cells with high side and forward scatter and high expression of SH antigens (> 95% of cells) by flow cytometry (Fig 4). There was no detectable difference in the staining of MSCs with the SH2, SH3, and SH4 MSC-specific antibodies after two passages versus six passages. There was no significant contamination of the MSC harvests with hematopoietic cells (CD45+ or CD14+). Detached MSCs appeared as large round cells (two to three times larger then neutrophils on a cytospin preparation) with a large nucleus and a lacy cytoplasm (Fig 5).



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  		Fig 4. Representative flow cytometric analysis of cultured MSCs with monoclonal (A) SH2, (B) SH3, (C) CD45, (D) SH4, and (E) CD14 antibodies. Black lines in each panel indicate isotype-matched mouse IgG antibody control staining. Human MSCs are stained strongly with SH2, SH3, and SH4, but not with CD45 and CD14.

 


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  		Fig 5. Photomicrograph of a detached MSC (magnification x100). After detachment, MSCs were mixed ex vivo with peripheral-blood mononuclear cells for direct comparison of size. Cytospin preparation (1,000 rpm) was made and a representative MSC was photographed along with admixed neutrophils.

 
In the last eight patients on the trial, MSCs were cryopreserved a median of 28.5 days (range, 20 to 30 days) after the start of culture. In these samples, viability after thawing determined by trypan blue staining was 84% ± 6%. Thawed MSCs were infused into patients within 10 minutes. A small aliquot of these cells were returned to in vitro cultures to analyze their proliferation. Approximately 1 x 106 thawed MSCs from all eight cryopreserved units reached greater than 90% confluence in a 175-cm2 flask within 7 days. This is the same duration required by 1 x 106 fresh MSCs to reach same degree of confluence.

Breast Cancer Contamination
At enrollment, four patients had breast cancer contamination in their marrow aspirate as determined by immunocytochemical analysis after staining with a cocktail of breast cancer–specific antibodies8 (Table 4). In two of these patients, cultured MSCs contained no detectable breast cancer cells and were infused into patients as described below. In the other two patients, breast cancer cells were detectable at first passage of MSCs in numbers 1 to 1.5 log lower than those found in the starting bone marrow aspirate. Nevertheless, these MSCs were not infused into these two patients as mandated by the clinical protocol.


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  		Table 4. Breast Cancer Cells in Bone Marrow Mononuclear Cells and MSC Cultures
 
PBPC and MSC Infusion
Patients received PBPC infusion containing a median of 13.9 x 106 CD34+ cells/kg (range, 1.5 to 39 x 106 CD34+ cells/kg). The 28 assessable patients also received 1 to 2.2 x 106 autologous MSCs/kg 1 or 24 hours after PBPC infusion. The MSC cell dose chosen was empiric and was influenced by safety consideration at the beginning of the study. On determination of safety, the dose was increased from 1 x 106 to 2 x 106 MSCs/kg. Patients whose MSCs were cryopreserved received 10% additional cells to compensate for potential cell loss during the cryopreservation procedure. The total number of MSCs infused was 51 to 174 x 106. Given that each confluent flask had 2.5 to 5 x 106 MSCs, it required harvesting a minimum of 10 flasks for all patients. In 15 patients, MSCs were given on the next day after PBPCs, and in the remaining 13 patients, MSC infusion occurred 1 hour after PBPCs (Table 2). In 20 patients, MSCs were harvested fresh on the day of infusion and were administered within 1 hour. In the remaining eight patients, a target dose of MSCs were harvested and cryopreserved, and when thawed, the cells were infused immediately. There was no immediate or delayed toxicity related to IV MSC infusion. None of the patients experienced allergic reactions or respiratory symptoms.

Detection of Clonogenic MSCs in Blood
MSCs were not detected in the blood at baseline in any patient. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion of autologous MSCs in 13 (62%) of 21 patients analyzed. MSCs that were recovered from the venous blood had morphology and surface marker expression (SH2, SH3, and SH4) that was identical to those recovered from bone marrow (Fig 6). Circulating MSCs were detected at the end of infusion in eight (38%) of 21 patients, 15 minutes later in 10 patients (48%), and 1 hour later in three patients (14%) (Fig 7). When seven patients who received 1.2 to 2.2 x 106 cryopreserved MSCs were analyzed separately, circulating MSC detection rates were 0% (0 of seven patients) before infusion, 85% (six patients) at the end of infusion, 43% (three patients) at 15 minutes, and 14% (one patient) at 1 hour.



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  		Fig 6. (A) Phase-contrast photomicrograph of MSCs isolated from blood immediately after infusion of autologous MSCs (magnification x100). Cellular morphology is similar to bone marrow–derived MSCs (Fig 1C). (B) Flow cytometric analysis of the same cells showing staining with SH-3 antibody. Open histogram indicates isotype control.

 


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  		Fig 7. Patients with detectable clonogenic MSCs in their venous blood after MSC infusion. Shown is the percentage of patients with evidence of circulating MSCs at indicated time points; n = number of patients analyzed at each time point.

 
Hematopoietic Engraftment and Clinical Outcome
Hematopoietic engraftment was prompt in all patients, with median neutrophil recovery (> 500/µL) in 8 days (range, 6 to 11 days) and platelet count recovery greater than 20,000/µL and greater than 50,000/µL unsupported in 8.5 days (range, 4 to 19 days) and 13.5 days (range, 7 to 44 days), respectively. Bone marrow CFU concentrations recovered to 70% of baseline by day 42 (Fig 8). All patients were discharged from the hospital. There was only one patient who died within the first 100 days of transplantation from unknown cause. This patient was evaluated on day T+21 and was free of symptoms and clinical findings. Median follow-up of the remaining patients is 9 months (range, 4 to 22 months). Three patients died as a result of disease progression. Of the 24 remaining patients, 11 are without evidence of disease, three have stable disease, and 10 have relapsed.



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  		Fig 8. Bone marrow hematopoietic CFU (CFU-granulocyte macrophage + erythroid burst-forming unit) concentrations (per 105 bone marrow mononuclear cells) before and after PBPC plus MSC infusion (arrow). Error bars represent one SD from the mean. Day 0 is the day of PBPC infusion.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report is the first describing that autologous MSCs can be successfully isolated, ex vivo culture-expanded, and infused IV without toxicity into advanced breast cancer patients at the time of PBPC transplantation. We have optimized MSC culture expansion methods to generate large numbers of autologous MSCs in a relatively short period of time for clinical use with a therapeutic intent. The culture technique was simple, yielding greater than 1 x 106 MSCs/kg patient weight in 3 to 4 weeks and could be efficiently carried out in a single institution without microbiologic contamination. Optimized culture conditions did not promote growth or survival of detectable contaminating breast cancer cells. In contrast to our earlier report,6 we infused autologous MSCs after myeloablative chemotherapy to promote survival and engraftment with PBPCs. Despite the MSCs large size and ex vivo culture with fetal calf serum–containing medium, there were no infusion-related immediate or delayed toxicities associated with administration of up to 2.2 x 106 MSCs/kg. Furthermore, hematopoietic reconstitution was rapid, particularly in platelet counts, in the majority of patients. Our results indicate that this form of novel cellular therapy is feasible and may have a number of beneficial clinical effects in the setting of hematopoietic stem-cell transplantation and should be studied in randomized trials.

MSCs seem to constitute an essential part of the marrow microenvironment and support hematopoiesis.5,12,13 A number of investigators have demonstrated that the bone marrow microenvironment is damaged because of the effects of alkylating agents and radiation, which diminishes its hematopoietic support function.14-18 We propose that culture-expanded MSCs can be used to improve the rate and quality of hematopoietic engraftment by regenerating the marrow microenvironment, particularly in patients who previously received stroma-damaging therapy.

Breast cancer patients treated with high-dose chemotherapy generally experience complete and rapid neutrophil and platelet engraftment when supported with mobilized PBPCs containing >= 2 x 106 CD34+ cells.19-21 On the other hand, patients receiving lower doses of CD34+ cells, or those undergoing tandem transplantations, are at increased risk for delayed platelet engraftment.22,23 Unsuccessful mobilization of CD34+ cells is commonly associated with extensive previous therapy and bone marrow metastasis, which are also associated with microenvironment damage. Coupled with low doses of CD34+ cells, abnormal marrow microenvironment increases the risk of delayed engraftment. Sequential use of high-dose chemotherapy is also toxic to the marrow microenvironment, as evidenced by the delayed hematopoietic recovery after the second transplantation despite infusion of an equal number of stem cells.22,23 Therefore, attempts to improve the bone marrow microenvironment are likely to improve hematopoiesis after myelotoxic treatment. We propose that infusion of autologous culture-expanded MSCs along with PBPCs may improve the bone marrow microenvironment and, subsequently, the rate and quality of hematopoietic recovery in heavily pretreated patients and those with bone and bone marrow metastasis. In addition, owing to their potential to differentiate into osteoblasts, human MSCs may play a role in the healing of metastatic bone lesions after high-dose chemotherapy, and this should be tested in further randomized clinical trials in patients with bone metastases. MSC-mediated new bone formation would have an important palliative effect in metastatic cancers by prevention of pathologic fractures. In other clinical situations when engraftment failure is high, such as in the case of HLA-mismatched sibling or matched unrelated donor marrow or umbilical cord-blood transplantation, autologous or allogeneic MSCs may decrease graft failure by facilitating engraftment and proliferation of hematopoietic precursors. In a murine allogeneic transplantation model, periosteum-derived osteoblasts were shown to promote engraftment of allogeneic hematopoietic stem cells, which suggests a supportive and immune regulatory role for mesenchymal cells.24

We recovered clonogenic MSCs from peripheral blood in 13 of 21 patients up to 60 minutes after IV infusion of MSCs. This observation indicates that these relatively large cells can traverse the circulation without loss of viability and proliferative capacity. None of the patients had circulating MSCs at baseline, which indicates that high-dose chemotherapy–related stromal injury does not promote circulation of endogenous MSCs. In addition, we have shown previously that MSCs do not circulate in peripheral blood during steady-state or after growth factor treatment, and, therefore, PBPC collections are devoid of MSCs.25 Demonstration of circulating clonogenic MSCs up to 60 minutes after infusion suggest that these cells can potentially distribute and survive in tissues.

Although culture-expanded MSCs can be safely infused into patients after high-dose chemotherapy, their distribution, survival, and participation in tissue function is largely unknown. Recipients of unmanipulated allogeneic bone marrow transplants were shown to regenerate their marrow stroma from autologous cells.26 These results were interpreted by the relative resistance of the stromal elements to myeloablative therapy, which allows regeneration of autologous stroma. In addition, the number of stromal precursors in the bone marrow graft is likely to be small, and the homing efficiency of these cells is unknown. Murine stromal cells were infused into mice by a number of investigators27-29 after radiation therapy and were found to facilitate hematopoietic recovery. Stromal cells of the COL1A1 transgenic mice were found 30 to 150 days later in marrow, spleen, bone, lung, and cartilage of syngeneic mice and constituted 1.5% to 12% of the cells.30 Similarly, genetically marked canine MSCs were infused into autologous as well as dog leukocyte antigen–identical litter-mate dogs after 9.2 Gy of total-body irradiation along with unmodified bone marrow or PBPCs.31,32 Green fluorescence protein gene–marked canine MSCs were found predominantly in the marrow of sternum, rib, and limbs at 6 and 14 weeks postinfusion. More recently, xenotransplantation models are being developed to determine the transplantability and homing of human MSCs in animals. Preliminary results show survival of human MSCs in immunocompromised mice33 and preimmune fetal sheep34 weeks after transplantation. A multicenter study investigating the safety of allogeneic culture-expanded MSC infusion in humans is currently underway, and genotypic differences between the donor and the recipient should allow us to determine the distribution of these cells in vivo. In addition, studies with marker- or therapeutic-gene transduced MSCs are being developed in the autologous setting to investigate distribution and homing of MSCs, as well as to use them as cellular vehicles for delivery of exogenous gene products.28

In summary, autologous MSCs can be isolated, rapidly expanded to large numbers, and infused into patients undergoing high-dose chemotherapy and autologous PBPC transplantation. Therapeutic potential of MSCs should be further investigated in the clinical setting.


   ACKNOWLEDGMENTS
 
Supported in part by Osiris Therapeutics Inc, Baltimore, MD, and Public Health Service grants no. MO1RR00080-35 (O.N.K) and P30CA43703.

We thank Dr Neelam Jaiswal, Guillermo Donate, and Robert M. Fox for their technical assistance, and Drs Annmarie Moseley and David Fink from Osiris Therapeutics Inc for their helpful discussions. Construction of the Cell and Gene Therapy Core Facility at the Case Western Reserve University was supported by a grant from Ohio Reagents.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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Submitted May 6, 1999; accepted August 11, 1999.




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