Engineering a Hematopoietic Stem Cell Niche By Revitalizing Mesenchymal Stem Cells with Five Transcription Factors
Authors: Nakahara Fumio , Pinho Sandra , Borger Daniel K. , Wei Qiaozhi , Maryanovich Maria , Zahalka Ali H. , Suzuki Masako , Cruz Cristian , Wang Zichen , Xu Chunliang , Boulais Philip , Ma'ayan Avi , Greally John M. , Frenette Paul S. 
Published: November 2019

Hematopoietic stem cells (HSCs) are maintained by bone marrow (BM) niches in vivo, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors (Scf, Cxcl12, Vcam1 and Angpt1) by Nestin-GFP+ mesenchymal-derived stem cells (MSCs) is downregulated upon culture and lose its effect of maintaining HSC in vitro, suggesting that transcriptional rewiring may contribute to this reduced potential in cultured MSCs.
To gain further insight, we searched RNA sequencing data for transcriptional regulators that were highly expressed in Nestin-GFP+ stroma, revealing 40 potential candidates. We compared the expression of these genes by real-time quantitative PCR (qPCR) in freshly isolated Nestin-GFP+ or Nestin-GFP- BM CD45-Ter119-CD31- cells, with that of cultured Nestin-GFP+ stroma. These analyses yielded 28 candidate genes after the elimination of 12 genes due to non-specific expression or lack of downregulation after culture. We cultured stromal cells isolated from Scf-GFP knock-in mice in which GFP expression reflects endogenous Scf mRNA synthesis. Upon culture, GFP expression was rapidly downregulated in these cells, demonstrating the potential of using GFP to screen for factors capable of revitalizing niche activity in cultured MSCs. We generated lentiviral vectors expressing 28 selected genes and transduced the viral mixture into cultured stromal cells derived from Scf-GFP mice. Five days after transduction, we observed re-emergence of GFP+ cells and these GFP+ cells were sorted and plated in limiting dilutions to isolate single cell-derived clones. Using this approach, we generated 16 independent GFP+ single cell-derived clones. To determine the specific combination of genes that enables cultured stromal cells to regain their capacity to maintain and expand HSCs in vitro, lineage-negative (Lin-) BM cells were co-cultured with each single cell-derived clone or control stroma. Thus, we identified 5 transcription factors (Klf7, Ostf1, Xbp1, Irf3, and Irf7; KOXII) that restored HSC niche function in cultured BM-derived MSCs.
These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs in vitro showed higher repopulation capacity and enabled lethally irradiated recipient mice to survive better. Competitive reconstitution assays revealed 7-fold expansion of functional HSCs by rMSCs. Moreover, rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress.
To investigate the revitalization mechanism, we performed ATAC-seq in freshly sorted Scf-GFP- CD45-Ter119-CD31- cells, Scf-GFP+ CD45-Ter119-CD31- cells, rMSCs and control vector-transduced stroma. We found that revitalization of MSCs led to 9,623 peaks of open chromatin in rMSCs when compared to control MSCs. Of these, 626 open peaks were also detected in freshly isolated Scf-GFP+ cells when compared to Scf-GFP- cells. Motif analyses of the sequence at these 626 peaks revealed that myocyte enhancer factor 2c (Mef2c) was among the most significantly enriched transcription regulators. Mef2c was also expressed at high levels in both rMSCs and freshly isolated Scf-GFP+ cells compared to control cultured MSCs and freshly isolated Scf-GFP- cells by RNA-seq and real-time qPCR. To evaluate the role of Mef2c in rMSCs, we knocked down Mef2c in rMSCs by short hairpin RNA lentiviral transduction (shMef2c). We found that the expression of niche factors (Scf, Cxcl12 and Vcam1) was reduced in shMef2c-transduced compared to parental rMSCs. In addition, shMef2c transduced-rMSCs exhibited reduced (by 43%) capacity to expand HSCs in co-culture compared to shCntrl transduced-rMSCs. These results suggest a role for Mef2c as a downstream effector mediating MSC revitalization. We are now exploring the method to make these rMSCs to form new niches in vivo.
Our results suggest that combination of KOXII genes are able to fully restore the niche activity in MSCs ex vivo and establish a new platform that provides critical insight in the regulatory network of the HSC niche leading to the basis toward the engineering of supportive niches for curative cell therapies.

Disclosures
Wei: Albert Einstein College of Medicine, Inc: Patents & Royalties. Frenette:Albert Einstein College of Medicine, Inc: Patents & Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.