Lineage mapping and characterization of the native progenitor population in cellular allograft

Abstract

Background context: The gold standard for bone grafting remains the autograft. However, the attractiveness of autograft is counterbalanced by donor site morbidity. To mimic autograft-and its fundamental properties of osteoconductivity, osteoinductivity, and osteogenicity-novel bone grafting materials such as cellular allograft (Osteocel Plus) are composed of allograft in which the progenitor cells are preserved. However, the true identity of these cells remains obscure largely due to the lack of specific bona fide antigenic markers for stem versus progenitor cells.

Purpose: To characterize the stem and progenitor population in cellular allograft, Osteocel Plus.

Study design: To determine whether cells endogenous to a cellular allograft undergo extensive self-renewal (a functional hallmark of stem cells), we employed a novel use of lineage mapping using a modern and refined replication incompetent lentiviral library with high complexity to uniquely label single cells with indelible genetic tags faithfully passed on to all progeny, allowing identification of highly proliferative clones. We used genetic and proteomic profiling as well as functional assays to show that these cells are capable of multipotential differentiation (the second functional hallmark of stem cells). Use of these two functional hallmarks enabled us to establish the existence of a stem and progenitor cell population in cellular allografts.

Methods: Specifically, we employed (1) cellular dissociation and (2) in vitro expansion and differentiation capacity of cells released from cellular allograft. We determined differential gene expression profiling of a bona fide human mesenchymal stem cell line and cells from cellular allograft using focused PCR arrays mesenchymal stem cell (MSC) and osteogenesis associated. Proteomic profiling of cells from cellular allograft was performed using (1) immunofluorescence for BMP-2, Runx2 SMADs, CD44, Stro-1, Collagen, RANKL, Osterix Osteocalcin, and Ki67; (2) flow cytometry for Ki67, CD44, Stro-1, Thy1, CD146, and Osteocalcin; and (3) enzyme-linked immunosorbent assays (ELISA) for BMP-2, Osteocalcin, RANKL, Osteoprotegrin, and Osteocalcin. Clonal analysis of cells from cellular allograft was performed utilizing advance lentivirus lineage mapping techniques and massive parallel sequencing. Alizarin Red, Alcian Blue, and Oil red O staining assessed tripotential differentiation capacity.

Results: Serial trypsinization of allograft cellular bone matrix yielded approximately 1×105 cells per mL with viability greater than 90%. Cells expressed a panel of 84 MSC-associated genes in a pattern similar to but not identical to pure MSCs; specifically, 59 of 84 genes showed less than a 2.5-fold change in both cell types. Protein analysis showed that cellular allograft -derived cells maintained in nondifferentiation media expressed the early osteo-progenitor markers BMP-2, SMADs, and Runx2. Corresponding flow cytometry data for MSC markers revealed the presence of Stro-1 (49%), CD44 (99%), CD90 (42%), and CD146 (97%). Lineage mapping indicated that 62% of clones persisted and generated progeny through 10 passages, strongly suggesting the presence of bona fide stem cells. Passage 10 clones also exhibited tri-lineage differentiation capacity into osteogenic (Alizarin Red with H&E counterstain), chondrogenic (Alcian Blue), and adipogenic (Oil red O). Cells that did not proliferate through 10 passages presumably differentiated along an osteo-progenitor lineage.

Conclusion: These data indicate that cellular allograft (Osteocel Plus) contains a heterogeneous population of cells with most cells demonstrating the capacity for extensive self-renewal and multipotential differentiation, which are hallmarks of stem cells. Whether stem cell-enriched allografts function comparably to autograft will require further studies, and their efficacy in facilitating arthrodesis will depend on randomized clinical studies.

Figure 1. Quantity, viability, and proliferative capacity of cells isolated from Osteocel Plus

(A) Cell yield and viability of 5ml of Osteocel Plus was determined by serial enzymatic digestion and Trypan blue exclusion staining (n=4). (B) Population growth curves used to calculate doubling time of isolated cells. (C) Immunoflourescent staining (10x) reveals proliferative capacity of cells released from Osteocel Plus. Phase contrast, Ki67 (APC, red), nuclear DAPI (blue) and merged images show that a high percentage of cells co-localize. (D) flow cytometry analysis for the proliferative marker Ki67; 97% of cells were gated (left panel) and 39% expressed Ki67 (right panel).

Figure 2. Migratory and proliferative capacity of Osteocel Plus cells in 3D culture

(A) Time-lapse imaging of cancellous bone (phase) and LOLIG-transduced cells (green). Migration of cells (broken circles) and proliferation (broken squares) were observed over 13.75 hours (Images taken at 20x). (B) Flow cytometry analysis for proliferative capacity (Ki67+) of LOLIG-positive cells.

Figure 3. Expression profiling of cells released from Osteocel Plus

(A) MSC-focused qPCR array results of Osteocel Plus cells versus huMSCs cultured for 7 days in vitro in MSC expansion media (top cluster panel). The Osteogenesis-focused qPCR array results of Osteocel Plus cells versus huMSCs cultured in osteogenic differentiation media for 28 days in vitro (bottom cluster panel). Red= increase in expression relative to huMSCs; Green= decrease in expression relative to huMSCs. (B) Relative expression of selected MSC genes from the MSC PCR array of Osteocel Plus cells compared to huMSCs. Both were cultured in MSC media 7 days prior to analysis. (C) Relative expression of osteogenesis-related genes BMP 2-7 (i-vi), alkaline phosphate (vii), Smad 1-4 (viii-xi), Runx2 (xii), Col1a1 (xiii), col4a3 (xiv), osteocalcin (xv) in MSC- and osteogenesis- focused qPCR arrays. Osteocel Plus cells were cultured in MSC and ODM media for 7 and 28 days in vitro, respectively (n=3, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; 95% Confidence interval of difference).

Figure 4. Proteomic profiling of Osteocel Plus cells cultured for 7 days in MSC media

(A) Immunofluorescent staining (20x magnification) for early stem and progenitor cell markers and transcription factors: Stro-1 (Alexa 488), CD44 (Cy3), Runx2 (Alexa 488), BMP2 (Cy3), SMAD 1,4 (Cy3), and nuclear DAPI (Blue). (B) Flow cytometry analysis for stem cell markers CD44, Stro-1, CD90/thy1, CD146, and the mature osteoblast marker Osteocalcin.

Figure 5. Proteomic profiling of Osteocel Plus cells cultured for 28 days in ODM

(A) Immunofluorescent staining (20x) for the osteoblast markers Collagen (Alexa 488), Integrin (Cy3), Osteocalcin (Cy3), Osterix (Cy3), RANKL (Cy3), and for nuclear DAPI (blue). (B) Flow cytometry analysis for CD254/RANKL. (C) ELISA and multiplex immunoassay for BMP-2, Osteocalcin, RANKL, Osteoprotegrin (OPG), and Osteopontin (OPN). Comparative analysis was performed on acellular cancellous bone, huMSCs, Osteocel Plus cells only, and native Osteocel Plus cells adherent to the bone. Concentrations are expressed as pg/ml. (n=3, *p<0.05, 95% Confidence interval of difference).

Figure 6. Lineage mapping clonal analysis and identification of stem cells from Osteocel Plus

(A) Passage 1 (P1) and 10 (P10) cells from Osteocel Plus transduced with LOLIG (Images taken at 10x). (B) Parallel sequencing of cells from P1 (blue) and P10 (red) demonstrating polyclonal population distribution of 100 random clones. (C) Tri-lineage differentiation potential of P10 stem cells into osteoblasts (Alizarin Red with H&E counterstain, left panel), chondrocytes (Alcian Blue, middle panel), and adipocytes (Oil red O, right panel) lineages (Images taken at 20x magnification).